MICROMIRs

ABSTRACT

The present invention relates to very short heavily modified oligonucleotides which target and inhibit microRNAs in vivo, and their use in medicaments and pharmaceutical compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. Nonprovisional applicationSer. No. 12/681,587, which is a National Stage of Application NumberPCT/DK2008/000344, filed Oct. 3, 2008, which claims the benefit of U.S.Provisional Application Nos. 61/028,062, filed Feb. 12, 2008,60/979,217, filed Oct. 11, 2007, and 60/977,497, filed Oct. 4, 2007; andEuropean Patent Application No. 08104780, filed Jul. 17, 2008, all ofwhich are incorporated herein by reference in their entireties.Furthermore we reference and incorporate by reference WO2007/112754 andWO2007/112753 which are earlier applications from the same applicants.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA EFS-WEB

The content of the electronically submitted sequence listing (Name:2763_(—)0130006_sequence_listing_ST25.txt; Size: 598,545 bytes; and Dateof Creation: Oct. 19, 2014) is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to very short oligonucleotides whichtarget and inhibit microRNAs in vivo, and their use in medicaments andpharmaceutical compositions.

BACKGROUND OF THE INVENTION

MicroRNAs (miRNAs) are an abundant class of short endogenous RNAs thatact as post-transcriptional regulators of gene expression bybase-pairing with their target mRNAs. They are processed from longer (ca70-80 nt) hairpin-like precursors termed pre-miRNAs by the RNAse IIIenzyme Dicer. MicroRNAs assemble in ribonucleoprotein complexes termedmiRNPs and recognize their target sites by antisense complementaritythereby mediating down-regulation of their target genes. Near-perfect orperfect complementarity between the miRNA and its target site results intarget mRNA cleavage, whereas limited complementarity between themicroRNA and the target site results in translational inhibition of thetarget gene.

A summary of the role of microRNAs in human diseases, and the inhibitionof microRNAs using single stranded oligonucleotides is provided byWO2007/112754 and WO2007/112753, which are both hereby incorporated byreference in its entirety. WO2008046911, hereby incorporated byreference, provides microRNA sequences which are associated with cancer.Numerous microRNAs have been associated with disease phenotypes and itis therefore desirable to provide substances capable of modulating theavailability of microRNAs in vivo. WO2007/112754 and WO2007/112753disclose short single stranded oligonucleotides which are considered toform a strong duplex with their target miRNA. SEQ ID NOs 1-45 areexamples of anti microRNA oligonucleotides as disclosed in WO2007/112754and WO2007/112753.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that the use of veryshort oligonucleotides which target microRNAs and which have a highproportion of nucleotide analogue nucleotides, such as LNA nucleotides,are highly effective in alleviating the repression of RNAs, such as anmRNA, by the targeted microRNAs in vivo.

The present invention provides an oligomer a contiguous sequence of 7,8, 9 or 10 nucleotide units in length, for use in reducing the effectiveamount of a microRNA target in a cell or an organism, wherein at least70%, such as at least 80% of the nucleotide units of the oligomer areselected from the group consisting of LNA units and 2′ substitutednucleotide analogues.

The present invention provides an oligomer a contiguous sequence of 7,8, 9 or 10 nucleotide units in length, for use in reducing the effectiveamount of a microRNA target in a cell or an organism, wherein at least70% of the nucleotide units of the oligomer are selected from the groupconsisting of LNA units and 2′ substituted nucleotide analogues, andwherein at least 50%, such as at least 60%, such as at least 70% of thenucleotide units of the oligomer are LNA units.

The invention provides oligomers of between 7-10 nucleotides in lengthwhich comprises a contiguous nucleotide sequence of a total of between7-10 nucleotides, such as 7, 8, 9, nucleotide units, wherein at least50% of the nucleotide units of the oligomer are nucleotide analogues.

The invention further provides for an oligomer of between 7-10nucleotides in length which comprises a contiguous nucleotide sequenceof a total of between 7-10 nucleotides, such as 7, 8, 9, or 10,nucleotide units, wherein the nucleotide sequence is complementary to acorresponding nucleotide sequence found in mammalian or viral microRNA,and wherein at least 50% of the nucleotide units of the oligomer arenucleotide analogues.

The present invention provides oligomers according to the invention as amedicament.

The present invention provides pharmaceutical compositions comprisingthe oligomer of the invention and a pharmaceutically acceptable diluent,carrier, salt or adjuvant.

The invention provides for a conjugate comprising an oligomer accordingto the invention, conjugated to at least one non-nucleotide orpolynucleotide entity, such as a sterol, such as cholesterol.

The invention provides for the use of an oligomer or a conjugateaccording to the invention, for the manufacture of a medicament for thetreatment of a disease or medical disorder associated with the presenceor over-expression of a microRNA, such as one or more of the microRNAsreferred to herein.

The invention provides for the treatment of a disease or medicaldisorder associated with the presence or overexpression of the microRNA,comprising the step of administering a composition (such as thepharmaceutical composition) comprising an oligomer or conjugateaccording to the invention to a patient suffering from or likely tosuffer from said disease or medical disorder.

The invention provides for a method for reducing the effective amount ofa microRNA target in a cell or an organism, comprising administering theoligomer of the invention, or a composition (such as a pharmaceuticalcomposition) comprising the oligomer or conjugate according to theinvention to the cell or organism.

The invention provides for a method for reducing the effective amount ofa microRNA target in a cell or an organism, comprising administering theoligomer or conjugate or pharmaceutical composition according to theinvention to the cell or organism.

The invention provides for a method for de-repression of a target mRNA(or one or more RNAs) in a cell or an organism, comprising administeringan oligomer or conjugate according to the invention, or a compositioncomprising said oligomer or conjugate, to said cell or organism.

The invention provides for the use of an oligomer or a conjugateaccording to the invention, for inhibiting the microRNA in a cell whichcomprises said microRNA, such as a human cell. The use may be in vivo orin vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic presentation of the miR-21, miR-155 and miR-122 8-merLNA-antimiRs (Compound 3205 (SEQ ID NO: 2); Compound 3207 (SEQ ID NO:4): Compound 3208 (SEQ ID NO: 6), indicating the targeting positionswith the fully LNA-modified and phosphorothiolated LNA-antimiR.Preferred hybridisation positions for 7-mer, 8-mer, 9-mer and 10-mer LNAoligonucleotides on the mature microRNA are also indicated.

FIG. 2. Assessment of miR-21 antagonism by Compound 3205 (SEQ ID NO: 2)and Compound 3204 (SEQ ID NO: 1) LNA-antimiRs in MCF-7 cells using aluciferase sensor assay. MCF-7 cells were co-transfected with luciferasesensor plasmids containing a perfect match target site for miR-21 or amismatch target site (.mm2) and LNA-antimiRs at differentconcentrations. After 24 hours, cells were harvested and luciferaseactivity measured. Shown are the mean of renilla/firefly ratios forthree separate experiments (bars=s.e.m), were all have been normalizedagainst 0 nM psiCHECK2 (=control).

FIG. 3. Assessment of miR-21 antagonism by Compound 3205 (SEQ ID NO: 2)and Compound 3204 (SEQ ID NO: 1) LNA-antimiRs in HeLa cells using aluciferase sensor assay. HeLa cells were co-transfected with luciferasesensor plasmids containing a perfect match target site for miR-21(mir-21) or a mismatch target site (mm2) and LNA-antimiRs at differentconcentrations. After 24 hours, cells were harvested and luciferaseactivity measured. Shown are the mean of renilla/firefly ratios forthree separate experiments (bars=s.e.m), were all have been normalizedagainst 0 nM psiCHECK2 (=control).

FIG. 4. Assessment of miR-155 antagonism by Compound 3206 (SEQ ID NO: 3)and Compound 3207 (SEQ ID NO: 4) LNA-antimiRs in LPS-treated mouse RAWcells using a luciferase sensor assay. RAW cells were co-transfectedwith miR-155 and the different LNA-antimiRs at different concentrations.After 24 hours, cells were harvested and luciferase activity measured.Shown are the mean of renilla/firefly, were all have been normalizedagainst 0 nM psiCHECK2.

FIG. 5. Assessment of miR-122 antagonism by Compound 3208 (SEQ ID NO: 6)and Compound 4 (SEQ ID NO: 5) LNA-antimiRs in HuH-7 cells using aluciferase sensor assay. HuH-7 cells were co-transfected with a miR-122luciferase sensor containing a perfect match miR-122 target site and thedifferent LNA-antimiRs at different concentrations. After 24 hours,cells were harvested and luciferase activity measured. Shown are themean of renilla/firefly ratios for three separate experiments(bars=s.e.m), where all have been normalized against 0 nM psiCHECK2(=control).

FIG. 6. Schematic presentation of the miR-21 luciferase reporterconstructs.

FIG. 7. Assessment of miR-21 antagonism by an 8-mer LNA-antimiR Compound3205 (SEQ ID NO: 2) versus a 15-mer LNA-antimiR Compound 3204 (SEQ IDNO: 1) in PC3 cells using a luciferase reporter assay. PC3 cells wereco-transfected with luciferase reporter plasmids containing a perfectmatch target site for miR-21 or a mismatch target site and LNA-antimiRsat different concentrations. After 24 hours, cells were harvested andluciferase activity measured. Shown are the mean values (bars=s.e.m) ofthree independent experiments where the renilla/firefly ratios have beennormalized against 0 nM empty vector without target site (=control).Shown is also a schematic presentation of the miR-21 sequence and thedesign and position of the LNA-antimiRs. LNA nucleotides are indicatedby ovals, and DNA residues are indicated by bars.

FIG. 8. Specificity assessment of miR-21 antagonism by an 8-merLNA-antimiR in HeLa cells using a luciferase reporter assay. HeLa cellswere co-transfected with luciferase reporter plasmids containing aperfect match or a mismatched target site for miR-21 and LNA-antimiRsCompound 3205 (SEQ ID NO: 2) or an 8-mer LNA mismatch control oligoCompound 3218 (SEQ ID NO: 16) at different concentrations. After 24hours, cells were harvested and luciferase activity was measured. Shownare the mean values (bars=s.e.m) for three independent experiments wherethe Renilla/firefly ratios have been normalized against 0 nM emptyvector without target site (=control). Shown is also a schematicpresentation of the miR-21 sequence and the design and position of theLNA-antimiRs. Mismatches are indicated by filled ovals.

FIG. 9. Assessment of the shortest possible length of a fullyLNA-modified LNA-antimiR that mediates effective antagonism of miR-21.HeLa cells were co-transfected with luciferase reporter plasmidscontaining a perfect match or a mismatch target site for miR-21 and theLNA-antimiRs at different concentrations (Compound 3209=6-mer (SEQ IDNO: 7) and Compound 3210=7-mer (SEQ ID NO: 8)). After 24 hours, cellswere harvested and luciferase activity measured. Shown are the meanvalues (bars=s.e.m) for three independent experiments where therenilla/firefly ratios have been normalized against 0 nM empty vectorwithout target site (=control). Shown is also a schematic presentationof the miR-21 sequence and the design and position of the LNA-antimiRs.

FIG. 10. Length assessment of fully LNA-substituted LNA-antimiRsantagonizing miR-21. HeLa cells were co-transfected with luciferasereporter plasmids containing a perfect match or a mismatch target sitefor miR-21 and LNA-antimiRs at different concentrations (Compound3211=9-mer (SEQ ID NO: 9), Compound 3212=10-mer (SEQ ID NO: 10),Compound 3213=12-mer (SEQ ID NO: 11) and Compound 3214=14-mer (SEQ IDNO: 12)). After 24 hours, cells were harvested and luciferase activitymeasured. Shown are the mean values (bars=s.e.m) for three independentexperiments where the renilla/firefly ratios have been normalizedagainst 0 nM empty vector without target site (=control). Shown is alsoa schematic presentation of the miR-21 sequence and the design andposition of the LNA-antimiRs.

FIG. 11. Determination of the most optimal position for an 8-merLNA-antimiR within the miR target recognition sequence. HeLa cells wereco-transfected with luciferase reporter plasmids containing a perfectmatch or a mismatch target site for miR-21 and the LNA-antimiRs atdifferent concentrations. After 24 hours, cells were harvested andluciferase activity measured. Shown are the mean values (bars=s.e.m) forthree independent experiments where the renilla/firefly ratios have beennormalized against 0 nM empty vector without target site (=control).Shown is also a schematic presentation of the miR-21 sequence and thedesign and position of the LNA-antimiRs (Compound 3205 (SEQ ID NO: 2;Compound 3215 (SEQ ID NO: 13); Compound 3216 (SEQ ID NO: 14); Compound3217 (SEQ ID NO: 15).

FIG. 12. Validation of interaction of the Pdcd4-3′-UTR and miR-21 by the8-mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR. HeLa cells wereco-transfected with a luciferase reporter plasmid containing part of the3′UTR of Pdcd4 gene and LNA-antimiRs at different concentrations(Compound 3205=8-mer, perfect match (SEQ ID NO: 2); Compound 3218=8-mer,mismatch (SEQ ID NO: 16); Compound 3204=15-mer, LNA/DNA mix (SEQ ID NO:1); Compound 3220=15-mer, gapmer (SEQ ID NO: 18)). After 24 hours, cellswere harvested and luciferase activity measured. Shown arerenilla/firefly ratios that have been normalized against 0 nM. Shown isalso a schematic presentation of the miR-21 sequence and the design andposition of the LNA-antimiRs.

FIG. 13. Comparison of an 8-mer LNA-antimiR (Compound 3207 (SEQ ID NO:4)) with a 15-mer LNA-antimiR (Compound 3206 (SEQ ID NO: 3)) inantagonizing miR-155 in mouse RAW cells. Mouse RAW cells wereco-transfected with luciferase reporter plasmids containing a perfectmatch for miR-155 and the different LNA-antimiRs at differentconcentrations. After 24 hours, cells were harvested and luciferaseactivity measured. Shown are the mean values (bars=s.e.m) of threeindependent experiments where the renilla/firefly ratios have beennormalized against 0 nM empty vector without miR-155 target site(=control). Shown is also a schematic presentation of the miR-155sequence and the design and position of the LNA-antimiRs.

FIG. 14. Assessment of c/EBPO □ Assessment of c/EBPer LNA antimiR(Compound 3207 (SEQ ID NO: 4)) with a 15-mer LNA-antimiR (Compound 3206(SEQ ID NO: 3)) in antagonizing miR-155 in mouse RAW cells. Mouse RAWcells were co-transfected with luciferase reporter plasmids containing aperfect match for miR-155 and the diffter 20 hours, cells were harvestedand western blot analysis of protein extracts from RAW cells wasperformed. The different isoforms of c/EBPβ are indicated, and theratios calculated on c/EBPβ LIP and beta-tubulin are shown below.

FIG. 15. Antagonism of miR-106b by a fully LNA-modified 8-mer (Compound3221 (SEQ ID NO: 19)) LNA-antimiR or by a 15-mer mixmer (Compound 3228(SEQ ID NO: 26)) antimiR. HeLa cells were co-transfected with luciferasereporter plasmids containing a perfect match for miR-106b and thedifferent LNA-antimiRs at different concentrations. After 24 hours,cells were harvested and luciferase activity measured. Shown are themean values of four replicates where the renilla/firefly ratios havebeen normalized against 0 nM empty vector without miRNA target site(=control). Shown is also a schematic presentation of the miR-106bsequence and the design and position of the LNA-antimiRs.

FIG. 16. Antagonism of miR-19b by a fully LNA-modified 8-mer (Compound3222 (SEQ ID NO: 20)) LNA-antimiR and a 15-mer (Compound 3229 (SEQ IDNO: 27)) mixmer antimiR. HeLa cells were co-transfected with luciferasereporter plasmids containing a perfect match for miR-19a and the twoLNA-antimiRs at different concentrations. After 24 hours, cells wereharvested and luciferase activity measured. Shown are the mean values offour replicate experiments, where the renilla/firefly ratios have beennormalized against 0 nM empty vector without a miR-19a target site(=control). Shown is also a schematic presentation of the miR-19asequence and the design and position of the LNA-antimiRs.

FIG. 17. Schematic presentation showing the mature human miR-221 (SEQ IDNO: 432) and miR-222 (SEQ ID NO: 434) sequences. Shown in the square isthe seed sequence (7-mer) that is conserved in both miRNA sequences.

FIG. 18. Targeting of a microRNA family using short, fullyLNA-substituted LNA-antimiR. PC3 cells were co-transfected withluciferase reporter plasmids for miR-221 and miR-222 separately ortogether and with the different LNA-antimiRs at varying concentrations.When co-transfecting with the LNA-antimiRs (15-mers) Compound 3223 (SEQID NO: 21) (against miR-221) and Compound 3224 (SEQ ID NO: 22) (againstmiR-222), the total concentration was 2 nM (1 nM each), whiletransfecting the cells with Compound 3225 (SEQ ID NO: 23) (7-mer) theconcentrations were 0, 1, 5, 10 or 25 nM. After 24 hours, cells wereharvested and luciferase activity measured. Shown are the mean values(bars=s.e.m) of three independent experiments where the renilla/fireflyratios have been normalized against 0 nM empty vector without a miRNAtarget site (=control). Shown is also a schematic presentation of themiR-221/222 sequence and the design and position of the LNA-antimiRs.

FIG. 19. Assessment of p27 protein levels as a functional readout forantagonism of the miR-221/222 family by the 7-mer Compound 3225 (SEQ IDNO: 23) LNA-antimiR. PC3 cells were transfected with the 7-merLNA-antimiR Compound 3225 (SEQ ID NO: 23) targeting both miR-221 andmiR-222 at varying concentrations. After 24 hours, cells were harvestedand protein levels were measured on a western blot. Shown are the ratiosof p27/tubulin.

FIG. 20. Assessment of miR-21 antagonism by an 8-mer LNA-antimiRCompound 3205 (SEQ ID NO: 2) versus a 15-mer LNA-antimiR Compound 3204(SEQ ID NO: 1) and an 8-mer with 2 mismatches Compound 3218 (SEQ ID NO:16) in HepG2 cells using a luciferase reporter assay.

HepG2 cells were co-transfected with luciferase reporter plasmidcontaining a perfect match target site for miR-21 and LNA-antimiRs atdifferent concentrations. After 24 hours, cells were harvested andluciferase activity measured. Shown are the mean values (bars=s.e.m) ofthree independent experiments where the renilla/firefly ratios have beennormalized against 0 nM empty vector without target site (=control).Shown is also a schematic presentation of the miR-21 sequence and thedesign and position of the LNA-antimiRs.

FIG. 21. Validation of interaction of the Pdcd4 3′UTR and miR-21 by the8-mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR versus the 15-merCompound 3204 (SEQ ID NO: 1) and an 8-mer with two mismatches Compound3218 (SEQ ID NO: 16).

Huh-7 cells were co-transfected with a luciferase reporter plasmidcontaining part of the 3′UTR of Pdcd4 gene, pre-miR-21 (10 nM) andLNA-antimiRs at different concentrations. After 24 hours, cells wereharvested and luciferase activity measured. Shown are the mean values(bars=s.e.m) of three independent experiments where the renilla/fireflyratios have been normalized against 0 nM empty vector without targetsite (=control). Shown is also a schematic presentation of the miR-21sequence and the design and position of the LNA-antimiRs.

FIG. 22. Antagonism of miR-21 by Compound 3205 (SEQ ID NO: 2) leads toincreased levels of Pdcd4 protein levels.

HeLa cells were transfected with 5 nM LNA-antimiR Compound 3205 (SEQ IDNO: 2) (perfect match), or Compound 3219 (SEQ ID NO: 17) LNA scrambled(8 mer) or Compound 3218 (SEQ ID NO: 16) (8-mer mismatch). Cells wereharvested after 24 hours and subjected to Western blot with Pdcd4antibody.

FIG. 23. ALT and AST levels in mice treated with Compound 3205 (SEQ IDNO: 2) (perfect match) or Compound 3218 (SEQ ID NO: 16) (mismatchcontrol). Mice were sacrificed after 14 days and after receiving 25mg/kg every other day.

FIG. 24. Assessment of PU.1 protein levels as a functional readout formiR-155 antagonism by short LNA-antimiR Compound 3207 (SEQ ID NO: 4).

THP-1 cells were co-transfected with pre-miR-155 (5 nmol) and differentLNA oligonucleotides (5 nM) and 100 ng/ml LPS was added. After 24 hours,cells were harvested and western blot analysis of protein extracts fromthe THP-1 cells was performed. PU.1 and tubulin are indicated.

FIG. 25. Assessment of p27 protein levels as a functional readout forantagonism of the miR-221/222 family by the 7-mer Compound 3225 (SEQ IDNO: 23) LNA-antimiR.

PC3 cells were transfected with the 7-mer LNA-antimiR Compound 3225 (SEQID NO: 23) targeting both miR-221 and miR-222 and a LNA scrambledcontrol at 5 and 25 nM. After 24 hours, cells were harvested and proteinlevels were measured on a western blot. Shown are the ratios ofp27/tubulin.

FIG. 26. Knock-down of miR-221/222 by the 7-mer Compound 3225 (SEQ IDNO: 23) (perfect match) LNA-antimiR reduces colony formation in softagar in PC3 cells.

PC3 cells were transfected with 25 nM of the 7-mer LNA-antimiR Compound3225 (SEQ ID NO: 23) targeting both miR-221 and miR-222 or a 7-merscrambled control Compound 3231 (SEQ ID NO: 28). After 24 hours, cellswere harvested and seeded on soft agar. After 12 days, colonies werecounted. One experiment has been done in triplicate.

FIG. 27. Overview of the human let-7 family, and of tested antagonists.

(upper) The sequences represent the mature miRNA for each member and thebox depicts nucleotides 2-16, the positions typically antagonized byLNA-antimiRs (let-7a, SEQ ID NO: 96; let-7b, SEQ ID NO: 98; let-7c, SEQID NO: 100; let-7d, SEQ ID NO: 102; let-7e, SEQ ID NO: 104; let-7f, SEQID NO: 106; let-7i, SEQ ID NO: 111; miR-98, SEQ ID NO: 947). Columns tothe right show the number of nucleotide differences compared to let-7a,within the seed (S: position 2-8), extended seed (ES; position 2-9), andthe remaining sequence typically targeted by LNA-antimiRs (NE; position9-16), respectively. Nucleotides with inverted colors are alteredcompared to let-7a. (lower) Summary of tested antagonists against thelet-7 family, including information on design, length and perfectlycomplementary targets. All compounds are fully phosphorothiolated.

FIG. 28. Assessment of let-7 antagonism by six different LNA-antimiRs inHuh-7 cells using a luciferase sensor assay.

Huh-7 cells were co-transfected with luciferase sensor plasmidscontaining a partial HMGA2 3′UTR (with four let-7 binding sites), withor without let-7a precursor (grey and black bars, respectively), andwith 6 different LNA-antimiRs at increasing concentrations. After 24hours, cells were harvested and luciferase activity measured. Shown arethe mean of renilla/firefly ratios for duplicate measurements andstandard deviations for each assay. Within each LNA-antimiR group allratios have been normalized to the average of wells containing no let-7aprecursor (black bars).

FIG. 29. Luciferase results from Huh-7 cells transfected with the HMGA23′UTR sensor plasmid, LNA-antimiRs Compound 3226 (SEQ ID NO: 24) (left)and Compound 3227 (SEQ ID NO: 25) (right), and pre-miRs for let-7a (A),let-7d (B), let-7e (C), and let-7i (D). Grey bars indicate the targetde-repression after pre-mis inclusion, whereas black control barsrepresent the equivalent level without pre-miR addition. Each ratio isbased on quadruplicate measurements and have been normalized against theaverage of wells containing no precursor (black bars) within eachtreatment group.

FIG. 30. Luciferase results from HeLa cells transfected with the HMGA23′UTR sensor plasmid or control vector, and the LNA-antimiR Compound3227 (SEQ ID NO: 25) at various concentrations. Each ratio is based onquadruplicate measurements normalized against untreated (0 nM) emptycontrol vector (psi-CHECK-2; grey bars).

FIG. 31. Assessment of miR-21 antagonism by 8 mer Compound 3205 (SEQ IDNO: 2) in HCT116 cells using a luciferase sensor assay. HCT116 cellswere co-transfected with luciferase sensor plasmids containing a perfectmatch target site for miR-21 (grey bars) and LNA-antimiR and controloligonucleotides at different concentrations. After 24 hours, cells wereharvested and luciferase activity measured. Shown is one typical exampleof two where the renilla/firefly ratios have been normalized against 0nM empty vector (=black bars).

FIG. 32. Silencing of miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2)LNA-antimiR reduces colony formation in soft agar in PC3 cells. PC3cells were transfected with 25 nM of the 8-mer LNA-antimiR Compound 3205(SEQ ID NO: 2) targeting miR-21. After 24 hours, cells were harvestedand seeded on soft agar. After 12 days, colonies were counted. Shown isthe mean of three separate experiments, each performed in triplicate,and normalised against 0 nM control (i.e. transfection but with no LNA).p=0.01898 for Compound 3205 (SEQ ID NO: 2).

FIG. 33. Knock-down of miR-21 by the 8-mer Compound 3205 (SEQ ID NO: 2)LNA-antimiR reduces colony formation in soft agar in HepG2 cells. HepG2cells were transfected with 25 nM of the 8-mer LNA-antimiR Compound 3205(SEQ ID NO: 2) targeting miR-21. After 24 hours, cells were harvestedand seeded on soft agar. After 17 days, colonies were counted. Shown isthe mean of three replicates from one experiment (bars=SEM).

FIG. 34A. Wound closure in the invasive human prostate cell line PC3after treatment with Compound 3205 (SEQ ID NO: 2). PC3 cells weretransfected at day 3 with LNA-antimiR and control oligonucleotides at 25nM, Compound 3205 (SEQ ID NO: 2) (8 mer, perfect match) and Compound3219 (SEQ ID NO: 17) (8 mer, mismatch) and the following day a scratchwas made. Pictures were taken after 24 hours in order to monitor themigration.

FIG. 34B. Histogram showing the area in each timepoint of FIG. 34Ameasured with the software program Image J and normalized againstrespective 0 h time-point.

FIG. 35. Length assessment of fully LNA-substituted LNA-antimiRsantagonizing miR-155. RAW cells were co-transfected with luciferasereporter plasmids containing a perfect match target site for miR-155 andwith LNA-antimiR oligonucleotides at different concentrations. After 24hours, cells were harvested and luciferase activity measured. Shown arethe mean values (bars=s.e.m) for three independent experiments where therenilla/firefly ratios have been normalized against 0 nM empty vectorwithout target site (=mock). Shown is also a schematic presentation ofthe miR sequence and the design and position of the LNA-antimiRs.

FIG. 36A. Binding of 5′-FAM labeled LNA-antimiR-21 Compound 3205 (SEQ IDNO: 2) to mouse plasma protein. The drawing shows % unboundLNA-antimiR-21 compound as a function of oligonucleotide concentrationin mouse plasma.

FIG. 36B. Binding of 5′-FAM labeled LNA-antimiR-21 Compound 3205 (SEQ IDNO: 2) to mouse plasma protein. The drawing shows concentration ofunbound LNA-antimiR-21 Compound 3205 (SEQ ID NO: 2) as a function ofCompound 3205 (SEQ ID NO: 2) concentration in mouse plasma.

FIG. 37. Quantification Ras protein levels by Western blot analysis. A.Gel image showing Ras and Tubulin (internal standard) protein in treated(anti-let-7; 8-mer) vs. untreated (saline) lung and kidney samples. B.Quantifications of Ras protein levels in the lung and kidney,respectively, of LNA-antimiR-treated mice (black bars), normalizedagainst equivalent saline controls (grey bars), using tubulin asequal-loading control.

FIG. 38. Silencing of miR-21 by Compound 3205 (SEQ ID NO: 2) leads toincreased levels of Pdcd4 protein levels in vivo. Mice were injectedwith saline or 25 mg/kg LNA-antimiR Compound 3205 (SEQ ID NO: 2) over 14days every other day, with a total of 5 doses. Mice were sacrificed andprotein was isolated from kidney and subjected to Western blot analysiswith Pdcd4 antibody. A. Gel image showing Pdcd4 and Gapdh (internalstandard) protein in treated (antimiR-21; 8-mer) vs. untreated (saline)kidney samples (M1, mouse 1; M2, mouse 2). B. Quantification of Pdcd4protein levels in kidneys of LNA-antimiR-treated mice (dark grey bars),normalized against the average of equivalent saline controls (light greybars), using Gapdh as loading control.

DETAILED DESCRIPTION OF THE INVENTION

Short oligonucleotides which incorporate LNA are known from the in vitroreagents area, (see for example WO2005/098029 and WO 2006/069584).However the molecules designed for diagnostic or reagent use are verydifferent in design than those for in vivo or pharmaceutical use. Forexample, the terminal nucleotides of the reagent oligos are typicallynot LNA, but DNA, and the internucleoside linkages are typically otherthan phosphorothioate, the preferred linkage for use in theoligonucleotides of the present invention. The invention thereforeprovides for a novel class of oligonucleotides (referred to herein asoligomers) per se.

The following embodiments refer to certain embodiments of the oligomerof the invention, which may be used in a pharmaceutical composition.Aspects which refer to the oligomer may also refer to the contiguousnucleotide sequence, and vice versa.

The Oligomer

The oligomer of the invention is a single stranded oligonucleotide whichcomprises nucleotide analogues, such as LNA, which form part of, or theentire contiguous nucleotide sequence of the oligonucleotide. Thenucleotide sequence of the oligomer consists of a contiguous nucleotidesequence.

The term “oligonucleotide” (or simply “oligo”), which is usedinterchangeably with the term “oligomer” refers, in the context of thepresent invention, to a molecule formed by covalent linkage of two ormore nucleotides. When used in the context of the oligonucleotide of theinvention (also referred to the single stranded oligonucleotide), theterm “oligonucleotide” may have, in one embodiment, for example havebetween 7-10 nucleotides, such as in individual embodiments, 7, 8, 9, or10.

The term ‘nucleotide’ refers to nucleotides, such as DNA and RNA, andnucleotide analogues. It should be recognised that, in some aspects, theterm nucleobase may also be used to refer to a nucleotide which may beeither naturally occurring or non-naturally occurring—in this respectthe term nucleobase and nucleotide may be used interchangeably herein.

In some embodiments, the contiguous nucleotide sequence consists of 7nucleotide analogues. In some embodiments, the contiguous nucleotidesequence consists of 8 nucleotide analogues. In some embodiments, thecontiguous nucleotide sequence consists of 9 nucleotide analogues.

In one embodiment at least about 50% of the nucleotides of the oligomerare nucleotide analogues, such as at least about 55%, such as at leastabout 60%, or at least about 65% or at least about 70%, such as at leastabout 75%, such as at least about 80%, such as at least about 85%, suchas at least about 90%, such as at least about 95% or such as 100%. Itwill also be apparent that the oligonucleotide may comprise of anucleotide sequence which consists of only nucleotide analogues.Suitably, the oligomer may comprise at least one LNA monomer, such as 2,3, 4, 5, 6, 7, 8, 9 or 10 LNA monomers. As described below, thecontiguous nucleotide sequence may consist only of LNA units (includinglinkage groups, such as phosphorothioate linkages), or may consist ofLNA and DNA units, or LNA and other nucleotide analogues. In someembodiments, the contiguous nucleotide sequence comprises either one ortwo DNA nucleotides, the remainder of the nucleotides being nucleotideanalogues, such as LNA unit.

In some embodiments, the contiguous nucleotide sequence consists of 6nucleotide analogues and a single DNA nucleotide. In some embodiments,the contiguous nucleotide consists of 7 nucleotide analogues and asingle DNA nucleotide. In some embodiments, the contiguous nucleotidesequence consists of 8 nucleotide analogues and a single DNA nucleotide.In some embodiments, the contiguous nucleotide sequence consists of 9nucleotide analogues and a single DNA nucleotide. In some embodiments,the contiguous nucleotide sequence consists of 7 nucleotide analoguesand two DNA nucleotides. In some embodiments, the contiguous nucleotidesequence consists of 8 nucleotide analogues and two DNA nucleotides.

The oligomer may consist of the contiguous nucleotide sequence.

In a specially preferred embodiment, all the nucleotide analogues areLNA. In a further preferred embodiment, all nucleotides of the oligomerare LNA. In a further preferred embodiment, all nucleotides of theoligomer are LNA and all internucleoside linkage groups arephosphothioate.

Herein, the term “nitrogenous base” is intended to cover purines andpyrimidines, such as the DNA nucleobases A, C, T and G, the RNAnucleobases A, C, U and G, as well as non-DNA/RNA nucleobases, such as5-methylcytosine (^(Me)C), isocytosine, pseudoisocytosine,5-bromouracil, 5-propynyluracil, 5-propyny-6-fluoroluracil,5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine,2,6-diaminopurine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanineand 2-chloro-6-aminopurine, in particular ^(Me)C. It will be understoodthat the actual selection of the non-DNA/RNA nucleobase will depend onthe corresponding (or matching) nucleotide present in the microRNAstrand which the oligonucleotide is intended to target. For example, incase the corresponding nucleotide is G it will normally be necessary toselect a non-DNA/RNA nucleobase which is capable of establishinghydrogen bonds to G. In this specific case, where the correspondingnucleotide is G, a typical example of a preferred non-DNA/RNA nucleobaseis ^(Me)C.

It should be recognised that the term in ‘one embodiment’ should notnecessarily be limited to refer to one specific embodiment, but mayrefer to a feature which may be present in ‘some embodiments’, or evenas a generic feature of the invention. Likewise, the use of the term‘some embodiments’ may be used to describe a feature of one specificembodiment, or a collection of embodiments, or even as a generic featureof the invention.

The terms “corresponding to” and “corresponds to” refer to thecomparison between the nucleotide sequence of the oligomer or contiguousnucleotide sequence (a first sequence) and the equivalent contiguousnucleotide sequence of a further sequence selected from either i) asub-sequence of the reverse complement of the microRNA nucleic acidtarget (such as a microRNA target selected from SEQ ID NO:40 to SEQ IDNO:976, and/or ii) the sequence of 9-mer, 8-mer and 7-mer nucleotidesprovided herein in Table 1. Nucleotide analogues are compared directlyto their equivalent or corresponding nucleotides. A first sequence whichcorresponds to a further sequence under i) or ii) typically is identicalto that sequence over the length of the first sequence (such as thecontiguous nucleotide sequence).

When referring to the length of a nucleotide molecule as referred toherein, the length corresponds to the number of monomer units, i.e.nucleotides, irrespective as to whether those monomer units arenucleotides or nucleotide analogues. With respect to nucleotides ornucleobases, the terms monomer and unit are used interchangeably herein.

It should be understood that when the term “about” is used in thecontext of specific values or ranges of values, the disclosure should beread as to include the specific value or range referred to.

As used herein, “hybridisation” means hydrogen bonding, which may beWatson-Crick, Hoogsteen, reversed Hoogsteen hydrogen bonding, etc.,between complementary nucleoside or nucleotide bases. The fournucleobases commonly found in DNA are G, A, T and C of which G pairswith C, and A pairs with T. In RNA T is replaced with uracil (U), whichthen pairs with A. The chemical groups in the nucleobases thatparticipate in standard duplex formation constitute the Watson-Crickface. Hoogsteen showed a couple of years later that the purinenucleobases (G and A) in addition to their Watson-Crick face have aHoogsteen face that can be recognised from the outside of a duplex, andused to bind pyrimidine oligonucleotides via hydrogen bonding, therebyforming a triple helix structure.

In the context of the present invention “complementary” refers to thecapacity for precise pairing between two nucleotides sequences with oneanother. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thecorresponding position of a DNA or RNA molecule, then theoligonucleotide and the DNA or RNA are considered to be complementary toeach other at that position. The DNA or RNA strand are consideredcomplementary to each other when a sufficient number of nucleotides inthe oligonucleotide can form hydrogen bonds with correspondingnucleotides in the target DNA or RNA to enable the formation of a stablecomplex. To be stable in vitro or in vivo the sequence of anoligonucleotide need not be 100% complementary to its target microRNA.The terms “complementary” and “specifically hybridisable” thus implythat the oligonucleotide binds sufficiently strong and specific to thetarget molecule to provide the desired interference with the normalfunction of the target whilst leaving the function of non-target RNAsunaffected. However, in one preferred embodiment the term complementaryshall mean 100% complementary or fully complementary.

In a preferred example the oligonucleotide of the invention is 100%complementary to a miRNA sequence, such as a human microRNA sequence, orone of the microRNA sequences referred to herein.

In a preferred example, the oligonucleotide of the invention comprises acontiguous sequence, which is 100% complementary to the seed region ofthe human microRNA sequence.

Preferably, the term “microRNA” or “miRNA”, in the context of thepresent invention, means an RNA oligonucleotide consisting of between 18to 25 nucleotides in length. In functional terms miRNAs are typicallyregulatory endogenous RNA molecules.

The terms “target microRNA” or “target miRNA” refer to a microRNA with abiological role in human disease, e.g. an upregulated, oncogenic miRNAor a tumor suppressor miRNA in cancer, thereby being a target fortherapeutic intervention of the disease in question.

The terms “target gene” or “target mRNA” refer to regulatory mRNAtargets of microRNAs, in which said “target gene” or “target mRNA” isregulated post-transcriptionally by the microRNA based on near-perfector perfect complementarity between the miRNA and its target siteresulting in target mRNA cleavage; or limited complementarity, oftenconferred to complementarity between the so-called seed sequence(nucleotides 2-7 of the miRNA) and the target site resulting intranslational inhibition of the target mRNA.

In the context of the present invention the oligonucleotide is singlestranded, this refers to the situation where the oligonucleotide is inthe absence of a complementary oligonucleotide—i.e. it is not a doublestranded oligonucleotide complex, such as an siRNA. In one embodiment,the composition according to the invention does not comprise a furtheroligonucleotide which has a region of complementarity with the oligomerof 5 or more, such as 6, 7, 8, 9, or 10 consecutive nucleotides, such aseight or more.

Length

Surprisingly we have found that such short ‘antimiRs’ provide animproved specific inhibition of microRNAs in vivo, whilst retainingremarkable specificity for the microRNA target. A further benefit hasbeen found to be the ability to inhibit several microRNAs simultaneouslydue to the conservation of homologous short sequences between microRNAspecies—such as the seed regions as described herein. According to thepresent invention, it has been found that it is particularlyadvantageous to have short oligonucleotides of 7, 8, 9, 10 nucleotides,such as 7, 8 or 9 nucleotides.

Sequences

The contiguous nucleotide sequence is complementary (such as 100%complementary—i.e. perfectly complementary) to a corresponding region ofa mammalian, human or viral microRNA (miRNA) sequence, preferably ahuman or viral miRNA sequence.

The microRNA sequence may suitably be a mature microRNA. In someembodiments the microRNA may be a microRNA precursor.

The human microRNA sequence may be selected from SEQ ID NO:1 to SEQ IDNO: 558 as disclosed in WO2008/046911, which are all hereby andspecifically incorporated by reference. As described in WO2008/046911,these microRNAs are associated with cancer.

The viral microRNA sequence may, in some embodiments, be selected fromthe group consisting of Herpes simplex virus 1, Kaposisarcoma-associated herpesvirus, Epstein Barr virus and Humancytomegalovirus.

In one embodiment, the contiguous nucleotide sequence is complementary(such as 100% complementary) to a corresponding region of a miRNAsequence selected from the group of miRNAs listed in table 1. Table 1provides 7-mer, 8-mer and 9-mer oligomers which target human and viralmicroRNAs published in miRBase (Release12.0—http://microrna.sanger.ac.uk/sequences/).

In some embodiments, the oligomers according to the invention mayconsist of or comprise a contiguous nucleotide sequence which iscomplementary to a corresponding microRNA sequence selected from thegroup consisting of miR-1, miR-10b, miR-17-3p, miR-18, miR-19a, miR-19b,miR-20, miR-21, miR-34a, miR-93, miR-106a, miR-106b, miR-122, miR-133,miR-134, miR-138, miR-155, miR-192, miR-194, miR-221, miR-222, miR-375.

Therefore, in one embodiment, the miRNA (i.e target miRNA) is selectedfrom the group consisting of miR-1, miR-10b, miR-17-3p, miR-18, miR-19a,miR-19b, miR-20, miR-21, miR-34a, miR-93, miR-106a, miR-106b, miR-122,miR-133, miR-134, miR-138, miR-155, miR-192, miR-194, miR-221, miR-222,and miR-375.

In one embodiment, the miRNA target is a member of the miR 17-92cluster, such as miR 17, miR 106a, miR 106b, miR 18, miR 19a, miR 19b/1,miR 19b/2, miR20/93, miR92/1, miR92/2 and miR25.

In some embodiments the contiguous nucleotide sequence is complementaryto a corresponding region of a microRNA (miRNA) sequence selected fromthe group consisting of miR-21, miR-155, miR-221, mir-222, and mir-122.

In some embodiments said miRNA is selected from the group consisting ofmiR-1, miR-10miR-29, miR-125b, miR-126, miR-133, miR-141, miR-143,miR-200b, miR-206, miR-208, miR-302, miR-372, miR-373, miR-375, andmiR-520c/e.

In some embodiments the contiguous nucleotide sequence is complementaryto a corresponding region of a microRNA (miRNA) sequence present in themiR 17-92 cluster, such as a microRNA selected from the group consistingof miR-17-5p, miR-20a/b, miR-93, miR-106a/b, miR-18a/b, miR-19a/b,miR-25, miR-92a, miR-363.

In one embodiment, the miRNA (i.e target miRNA) is miR-21, such ashsa-miR-21 (SEQ ID NO: 410). In one embodiment, the miRNA (i.e targetmiRNA) is miR-122, such as hsa-miR-122 (SEQ ID NO: 150). In oneembodiment, the miRNA (i.e target miRNA) is miR-19b, such as hsa-miR-19b(SEQ ID NO: 389). In one embodiment, the miRNA (i.e target miRNA) ismiR-155, such as hsa-miR-155 (SEQ ID NO: 355). In one embodiment, themiRNA (i.e target miRNA) is miR-375, such as hsa-miR-375 (SEQ ID NO:561). In one embodiment, the miRNA (i.e target miRNA) is miR-375, suchas hsa-miR-106b (SEQ ID NO: 124).

Suitably, the contiguous nucleotide sequence may be complementary to acorresponding region of the microRNA, such as a hsa-miR selected fromthe group consisting of 19b (SEQ ID NO: 389), 21 (SEQ ID NO: 410), 122(SEQ ID NO: 150), 155 (SEQ ID NO: 355) and 375 (SEQ ID NO: 561).

The Seed Region and Seedmers

The inventors have found that carefully designed short single strandedoligonucleotides comprising or consisting of nucleotide analogues, suchas high affinity nucleotide analogues such as locked nucleic acid (LNA)units, show significant silencing of microRNAs, resulting in reducedmicroRNA levels. It was found that tight binding of saidoligonucleotides to the so-called seed sequence, typically nucleotides 2to 8 or 2 to 7, counting from the 5′ end, of the target microRNAs wasimportant. Nucleotide 1 of the target microRNAs is a non-pairing baseand is most likely hidden in a binding pocket in the Ago 2 protein.Whilst not wishing to be bound to a specific theory, the presentinventors consider that by selecting the seed region sequences,particularly with oligonucleotides that comprise LNA, preferably LNAunits in the region which is complementary to the seed region, theduplex between miRNA and oligonucleotide is particularly effective intargeting miRNAs, avoiding off target effects, and possibly providing afurther feature which prevents RISC directed miRNA function.

The inventors have found that microRNA silencing is even more enhancedwhen LNA-modified single stranded oligonucleotides do not contain anucleotide at the 3′ end corresponding to this non-paired nucleotide 1.It was further found that at least two LNA units in the 3′ end of theoligonucleotides according to the present invention made saidoligonucleotides highly nuclease resistant.

In one embodiment, the first or second 3′ nucleotide of the oligomercorresponds to the second 5′ nucleotide of the microRNA sequence, andmay be a nucleotide analogue, such as LNA.

In one embodiment, nucleotide units 1 to 6 (inclusive) of the oligomeras measured from the 3′ end the region of the oligomer are complementaryto the microRNA seed region sequence, and may all be nucleotideanalogues, such as LNA.

In one embodiment, nucleotide units 1 to 7 (inclusive) of the oligomeras measured from the 3′ end the region of the oligomer are complementaryto the microRNA seed region sequence, and may all be nucleotideanalogues, such as LNA.

In one embodiment, nucleotide units 2 to 7 (inclusive) of the oligomeras measured from the 3′ end the region of the oligomer are complementaryto the microRNA seed region sequence, and may all be nucleotideanalogues, such as LNA.

In one embodiment, the oligomer comprises at least one nucleotideanalogue unit, such as at least one LNA unit, in a position which iswithin the region complementary to the miRNA seed region. The oligomermay, in one embodiment comprise at between one and 6 or between 1 and 7nucleotide analogue units, such as between 1 and 6 and 1 and 7 LNAunits, in a position which is within the region complementary to themiRNA seed region.

In one embodiment, the contiguous nucleotide sequence consists of orcomprises a sequence which is complementary (such as 100% complementary)to the seed sequence of said microRNA.

In one embodiment, the contiguous nucleotide sequence consists of orcomprises a sequence selected from any one of the seedmer sequenceslisted in table 1.

In one embodiment, the 3′ nucleotide of the seedmer forms the 3′ mostnucleotide of the contiguous nucleotide sequence, wherein the contiguousnucleotide sequence may, optionally, comprise one or two furthernucleotide 5′ to the seedmer sequence.

In one embodiment, the oligomer does not comprise a nucleotide whichcorresponds to the first nucleotide present in the microRNA sequencecounted from the 5′ end.

In one embodiment, the oligonucleotide according to the invention doesnot comprise a nucleotide at the 3′ end that corresponds to the first 5′end nucleotide of the target microRNA.

Nucleotide Analogues

According to the present invention, it has been found that it isparticularly advantageous to have short oligonucleotides of 7, 8, 9, 10nucleotides, such as 7, 8 or 9 nucleotides, wherein at least 50%, suchas 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or such as 100% of thenucleotide units of the oligomer are (preferably high affinity)nucleotide analogues, such as a Locked Nucleic Acid (LNA) nucleotideunit.

In some embodiments, the oligonucleotide of the invention is 7, 8 or 9nucleotides long, and comprises a contiguous nucleotide sequence whichis complementary to a seed region of a human or viral microRNA, andwherein at least 75%, such as at least 80%, such as at least 85%, suchas at least 90%, such as at least 95%, such as 100% of the nucleotidesare Locked Nucleic Acid (LNA) nucleotide units.

In such oligomers, in some embodiments, the linkage groups are otherthan phosphodiester linkages, such as are phosphorothioate linkages.

In one embodiment, all of the nucleotide units of the contiguousnucleotide sequence are LNA nucleotide units.

In one embodiment, the contiguous nucleotide sequence comprises orconsists of 7, 8, 9 or 10, preferably contiguous. LNA nucleotide units.

In a further preferred embodiment, the oligonucleotide of the inventionis 7, 8 or 9 nucleotides long, and comprises a contiguous nucleotidesequence which is complementary to a seed region of a human or viralmicroRNA, and wherein at least 80% of the nucleotides are LNA, andwherein at least 80%, such as 85%, such as 90%, such as 95%, such as100% of the internucleotide bonds are phosphorothioate bonds. It will berecognised that the contiguous nucleotide sequence of the oligomer (aseedmer) may extend beyond the seed region.

In some embodiments, the oligonucleotide of the invention is 7nucleotides long, which are all LNA.

In some embodiments, the oligonucleotide of the invention is 8nucleotides long, of which up to 1 nucleotide may be other than LNA. Insome embodiments, the oligonucleotide of the invention is 9 nucleotideslong, of which up to 1 or 2 nucleotides may be other than LNA. In someembodiments, the oligonucleotide of the invention is 10 nucleotideslong, of which 1, 2 or 3 nucleotides may be other than LNA. Thenucleotides ‘other than LNA, may for example, be DNA, or a 2′substituted nucleotide analogues.

High affinity nucleotide analogues are nucleotide analogues which resultin oligonucleotides which has a higher thermal duplex stability with acomplementary RNA nucleotide than the binding affinity of an equivalentDNA nucleotide. This may be determined by measuring the T_(m).

In some embodiments, the nucleotide analogue units present in thecontiguous nucleotide sequence are selected, optionally independently,from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit,2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INAunit, and a 2′MOE RNA unit.

In some embodiments, the nucleotide analogue units present in thecontiguous nucleotide sequence are selected, optionally independently,from the group consisting of 2′-O_alkyl-RNA unit, 2′-OMe-RNA unit,2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, and a 2′MOE RNA unit.

The term 2′fluoro-DNA refers to a DNA analogue with a substitution tofluorine at the 2′ position (2′F). 2′fluoro-DNA is a preferred form of2fluoro-nucleotide.

In some embodiments, the oligomer comprises at least 4 nucleotideanalogue units, such as at least 5 nucleotide analogue units, such as atleast 6 nucleotide analogue units, such as at least 7 nucleotideanalogue units, such as at least 8 nucleotide analogue units, such as atleast 9 nucleotide analogue units, such as 10, nucleotide analogueunits.

In one embodiment, the oligomer comprises at least 3 LNA units, such asat least 4 LNA units, such as at least 5 LNA units, such as at least 6LNA units, such as at least 7 LNA units, such as at least 8 LNA units,such as at least 9 LNA units, such as 10 LNA.

In one embodiment wherein at least one of the nucleotide analogues, suchas LNA units, is either cytosine or guanine, such as between 1-10 of theof the nucleotide analogues, such as LNA units, is either cytosine orguanine, such as 2, 3, 4, 5, 6, 7, 8, or 9 of the of the nucleotideanalogues, such as LNA units, is either cytosine or guanine.

In one embodiment at least two of the nucleotide analogues such as LNAunits are either cytosine or guanine. In one embodiment at least threeof the nucleotide analogues such as LNA units are either cytosine orguanine. In one embodiment at least four of the nucleotide analoguessuch as LNA units are either cytosine or guanine. In one embodiment atleast five of the nucleotide analogues such as LNA units are eithercytosine or guanine. In one embodiment at least six of the nucleotideanalogues such as LNA units are either cytosine or guanine. In oneembodiment at least seven of the nucleotide analogues such as LNA unitsare either cytosine or guanine. In one embodiment at least eight of thenucleotide analogues such as LNA units are either cytosine or guanine.

In a preferred embodiment the nucleotide analogues have a higher thermalduplex stability for a complementary RNA nucleotide than the bindingaffinity of an equivalent DNA nucleotide to said complementary RNAnucleotide.

In one embodiment, the nucleotide analogues confer enhanced serumstability to the single stranded oligonucleotide.

Whilst the specific SEQ IDs in the sequence listing and table 1 refer tooligomers of LNA monomers with phosphorothioate (PS) backbone, it willbe recognised that the invention also encompasses the use of othernucleotide analogues and/or linkages, either as an alternative to, or incombination with LNA. As such, the sequence of nucleotides (bases) shownin the sequence listings may be of LNA such as LNA/PS, LNA or may beoligomers containing alternative backbone chemistry, such assugar/linkage chemistry, whilst retaining the same base sequence (A, T,C or G).

Whilst it is envisaged that other nucleotide analogues, such as 2′-MOERNA or 2′-fluoro nucleotides may be useful in the oligomers according tothe invention, it is preferred that the oligomers have a highproportion, such as at least 50%, LNA, nucleotides. The nucleotideanalogue may be a DNA analogue such as a DNA analogue where the 2′-Hgroup is substituted with a substitution other than —OH (RNA) e.g. bysubstitution with —O—CH₃, —O—CH₂—CH₂—O—CH₃, —O—CH₂—CH—CH₂—NH₂,—O—CH₂—CH₂—CH₂—OH or —F. The nucleotide analogue may be a RNA analoguessuch as a RNA analogue which have been modified in its 2′-OH group, e.g.by substitution with a group other than —H (DNA), for example —O—CH₃,—O—CH₂—CH₂—O—CH₃, —O—CH₂—CH₂—CH₂—NH₂, —O—CH₂—CH₂—CH₂—OH or —F. In oneembodiment the nucleotide analogue is “ENA”.

LNA

When used in the present context, the terms “LNA unit”, “LNA monomer”,“LNA residue”, “locked nucleic acid unit”, “locked nucleic acid monomer”or “locked nucleic acid residue”, refer to a bicyclic nucleosideanalogue. LNA units are described in inter alia WO 99/14226, WO00/56746, WO 00/56748, WO 01/25248, WO 02/28875, WO 03/006475 and WO03/095467. The LNA unit may also be defined with respect to its chemicalformula. Thus, an “LNA unit”, as used herein, has the chemical structureshown in Scheme 1 below:

wherein

-   -   X is selected from the group consisting of O, S and NR^(H),        where R^(H) is H or C₁₋₄-alkyl; Y is (—CH₂)_(r), where r is an        integer of 1-4; and B is a nitrogenous base.

In a preferred embodiment of the invention, r is 1 or 2, in particular1, i.e. a preferred LNA unit has the chemical structure shown in Scheme2 below:

wherein X and B are as defined above.

In an interesting embodiment, the LNA units incorporated in theoligonucleotides of the invention are independently selected from thegroup consisting of thio-LNA units, amino-LNA units and oxy-LNA units.

Thus, the thio-LNA unit may have the chemical structure shown in Scheme3 below:

wherein B is as defined above.

Preferably, the thio-LNA unit is in its beta-D-form, i.e. having thestructure shown in 3A above. likewise, the amino-LNA unit may have thechemical structure shown in Scheme 4 below:

wherein B and R^(H) are as defined above.

Preferably, the amino-LNA unit is in its beta-D-form, i.e. having thestructure shown in 4A above.

The oxy-LNA unit may have the chemical structure shown in Scheme 5below:

wherein B is as defined above.

Preferably, the oxy-LNA unit is in its beta-D-form, i.e. having thestructure shown in 5A above. As indicated above, B is a nitrogenous basewhich may be of natural or non-natural origin. Specific examples ofnitrogenous bases include adenine (A), cytosine (C), 5-methylcytosine(^(Me)C), isocytosine, pseudoisocytosine, guanine (G), thymine (T),uracil (U), 5-bromouracil, 5-propynyluracil, 5-propyny-6,5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine,2,6-diaminopurine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanineand 2-chloro-6-aminopurine.

The term “thio-LNA unit” refers to an LNA unit in which X in Scheme 1 isS. A thio-LNA unit can be in both the beta-D form and in the alpha-Lform. Generally, the beta-D form of the thio-LNA unit is preferred. Thebeta-D-form and alpha-L-form of a thio-LNA unit are shown in Scheme 3 ascompounds 3A and 3B, respectively.

The term “amino-LNA unit” refers to an LNA unit in which X in Scheme 1is NH or NR^(H), where R^(H) is hydrogen or C₁₋₄-alkyl. An amino-LNAunit can be in both the beta-D form and in the alpha-L form. Generally,the beta-D form of the amino-LNA unit is preferred. The beta-D-form andalpha-L-form of an amino-LNA unit are shown in Scheme 4 as compounds 4Aand 4B, respectively.

The term “oxy-LNA unit” refers to an LNA unit in which X in Scheme 1 isO. An Oxy-LNA unit can be in both the beta-D form and in the alpha-Lform. Generally, the beta-D form of the oxy-LNA unit is preferred. Thebeta-D form and the alpha-L form of an oxy-LNA unit are shown in Scheme5 as compounds 5A and 5B, respectively.

In the present context, the term “C₁₋₆-alkyl” is intended to mean alinear or branched saturated hydrocarbon chain wherein the longestchains has from one to six carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl,isopentyl, neopentyl and hexyl. A branched hydrocarbon chain is intendedto mean a C₁₋₆-alkyl substituted at any carbon with a hydrocarbon chain.

In the present context, the term “C₁₋₄-alkyl” is intended to mean alinear or branched saturated hydrocarbon chain wherein the longestchains has from one to four carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Abranched hydrocarbon chain is intended to mean a C₁₋₄-alkyl substitutedat any carbon with a hydrocarbon chain.

When used herein the term “C₁₋₆-alkoxy” is intended to meanC₁₋₈-alkyl-oxy, such as methoxy, ethoxy, n-propoxy, isopropoxy,n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy,neopentoxy and hexoxy.

In the present context, the term “C₂₋₆-alkenyl” is intended to mean alinear or branched hydrocarbon group having from two to six carbon atomsand containing one or more double bonds. Illustrative examples ofC₂₋₆-alkenyl groups include allyl, homo-allyl, vinyl, crotyl, butenyl,butadienyl, pentenyl, pentadienyl, hexenyl and hexadienyl. The positionof the unsaturation (the double bond) may be at any position along thecarbon chain.

In the present context the term “C₂₋₆-alkynyl” is intended to meanlinear or branched hydrocarbon groups containing from two to six carbonatoms and containing one or more triple bonds. Illustrative examples ofC₂₋₆-alkynyl groups include acetylene, propynyl, butynyl, pentynyl andhexynyl. The position of unsaturation (the triple bond) may be at anyposition along the carbon chain. More than one bond may be unsaturatedsuch that the “C₂₋₆-alkynyl” is a di-yne or enedi-yne as is known to theperson skilled in the art.

When referring to substituting a DNA unit by its corresponding LNA unitin the context of the present invention, the term “corresponding LNAunit” is intended to mean that the DNA unit has been replaced by an LNAunit containing the same nitrogenous base as the DNA unit that it hasreplaced, e.g. the corresponding LNA unit of a DNA unit containing thenitrogenous base A also contains the nitrogenous base A. The exceptionis that when a DNA unit contains the base C, the corresponding LNA unitmay contain the base C or the base m C, preferably ^(Me)C.

Herein, the term “non-LNA unit” refers to a nucleoside different from anLNA-unit. i.e. the term “non-LNA unit” includes a DNA unit as well as anRNA unit. A preferred non-LNA unit is a DNA unit.

The terms “unit”, “residue” and “monomer” are used interchangeablyherein.

The term “at least one” encompasses an integer larger than or equal to1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19.20 and so forth.

The terms “a” and “an” as used about a nucleotide, an agent, an LNAunit, etc., is intended to mean one or more. In particular, theexpression “a component (such as a nucleotide, an agent, an LNA unit, orthe like) selected from the group consisting of . . . ” is intended tomean that one or more of the cited components may be selected. Thus,expressions like “a component selected from the group consisting of A, Band C” is intended to include all combinations of A. B and C, i.e. A, B,C, A+B, A+C, B+C and A+B+C.

Internucleoside Linkages

The term “internucleoside linkage group” is intended to mean a groupcapable of covalently coupling together two nucleotides, such as betweenDNA units, between DNA units and nucleotide analogues, between twonon-LNA units, between a non-LNA unit and an LNA unit, and between twoLNA units, etc. Examples include phosphate, phosphodiester groups andphosphorothioate groups.

In some embodiments, at least one of, such as all of the internucleosidelinkage in the oligomer is phosphodiester. However for in vivo use,phosphorothioate linkages may be preferred.

Typical internucleoside linkage groups in oligonucleotides are phosphategroups, but these may be replaced by internucleoside linkage groupsdiffering from phosphate. In a further interesting embodiment of theinvention, the oligonucleotide of the invention is modified in itsinternucleoside linkage group structure, i.e. the modifiedoligonucleotide comprises an internucleoside linkage group which differsfrom phosphate. Accordingly, in a preferred embodiment, theoligonucleotide according to the present invention comprises at leastone internucleoside linkage group which differs from phosphate.

Specific examples of internucleoside linkage groups which differ fromphosphate (—O—P(O)₂—O—) include —O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—,—S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—, —S—P(O)₂—S—,—O—PO(R^(H))—O—, —O—PO(OCH₃)—O—, —O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—,—O—PO(BH₃)—O—, —O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—,—NR^(H)—CO—O—, —NR^(H)—CO—NR^(H)—, —O—CO—O—, —O—CO—NR^(H)—,—NR^(H)—CO—CH₂—, —O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CO—NR^(H)—CH₂—,—CH₂—NR^(H)—CO—, —O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—,—CH₂—SO₂—CH₂, —CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—CO—, —CH₂—NCH₃—OCH₂—,where R^(H) is hydrogen or C₁₋₄-alkyl.

When the internucleoside linkage group is modified, the internucleosidelinkage group is preferably a phosphorothioate group (—O—P(O,S)—O—). Ina preferred embodiment, all internucleoside linkage groups of theoligonucleotides according to the present invention arephosphorothioate.

The internucleoside linkage may be selected form the group consistingof: —O—P(O)₂—O—, —O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—,—S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—,—O—PO(OCH₃)—O—, —O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,—NR^(H)—CO—NR^(H)—, and/or the internucleoside linkage may be selectedform the group consisting of: —O—CO—O—, —O—CO—NR^(H)—, —NR^(H)—CO—CH₂—,—O—CH₂—CO—NR^(H)—, —O—CH₂—CHNR^(H)—, —CO—NR^(H)—CH₂—, —CH₂—NR^(H)—CO—,—O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—SO₂—CH₂—,—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—CO—, —CH₂—NCH₃—O—CH₂—, where R^(H) isselected from hydrogen and C₁₋₄-alkyl. Suitably, in some embodiments,sulphur (S) containing internucleoside linkages as provided above may bepreferred. The internucleoside linkages may be independently selected,or all be the same, such as phosphorothioate linkages.

In one embodiment, at least 75%, such as 80% or 85% or 90% or 95% or allof the internucleoside linkages present between the nucleotide units ofthe contiguous nucleotide sequence are phosphorothioate internucleosidelinkages.

Micromir Oligonucleotides Targeting More than One microRNA

In one embodiment, the contiguous nucleotide sequence is complementaryto the corresponding sequence of at least two miRNA sequences such as 2,3, 4, 5, 6, 7, 8, 9, or 10 miRNA sequence. The use of a single universalbase may allow a single oligomer of the invention to target twoindependent microRNAs which either one or both have a single mismatch inthe region which corresponds to oligomer at the position where theuniversal nucleotide is positioned.

In one embodiment, the contiguous nucleotide sequence consists of orcomprises a sequence which is complementary to the sequence of at leasttwo miRNA seed region sequences such as 2, 3, 4, 5, 6, 7, 8, 9, or 10miRNA seed region sequences.

In one embodiment, the contiguous nucleotide sequence is complementaryto the corresponding region of both miR-221 and miR-222.

In one embodiment, the contiguous nucleotide sequence is complementaryto the corresponding region of more than one member of the miR-17-92duster—such as two or more or all of miR-17-5p, miR-20a/b, miR-93,miR-106a/b; or two or more or all of miR-25, miR-92a and miR-363.

In one embodiment, the contiguous nucleotide sequence consists of orcomprises a sequence that is complementary to 5′GCTACAT3′.

Oligomer Design

In one embodiment, the first nucleotide of the oligomer according to theinvention, counting from the 3′ end, is a nucleotide analogue, such asan LNA unit. In one embodiment, which may be the same or different, thelast nucleotide of the oligomer according to the invention, countingfrom the 3′ end, is a nucleotide analogue, such as an LNA unit.

In one embodiment, the second nucleotide of the oligomer according tothe invention, counting from the 3′ end, is a nucleotide analogue, suchas an LNA unit.

In one embodiment, the ninth and/or the tenth nucleotide of the oligomeraccording to the invention, counting from the 3′ end, is a nucleotideanalogue, such as an LNA unit.

In one embodiment, the ninth nucleotide of the oligomer according to theinvention, counting from the 3′ end is a nucleotide analogue, such as anLNA unit.

In one embodiment, the tenth nucleotide of the oligomer according to theinvention, counting from the 3′ end is a nucleotide analogue, such as anLNA unit.

In one embodiment, both the ninth and the tenth nucleotide of theoligomer according to the invention, calculated from the 3′ end is anucleotide analogue, such as an LNA unit.

In one embodiment, the oligomer according to the invention does notcomprise a region of more than 3 consecutive DNA nucleotide units. Inone embodiment, the oligomer according to the invention does notcomprise a region of more than 2 consecutive DNA nucleotide units.

In one embodiment, the oligomer comprises at least a region consistingof at least two consecutive nucleotide analogue units, such as at leasttwo consecutive LNA units.

In one embodiment, the oligomer comprises at least a region consistingof at least three consecutive nucleotide analogue units, such as atleast three consecutive LNA units.

Other Patterns of Nucleotide Analogues Such as LNA in the Oligomer

Whilst it is envisaged that oligomers containing at least 6 LNA, such asat least 7 nucleotide units may be preferable, the discovery that suchshort oligomers are highly effective at targeting microRNAs in vivo canbe used to prepare shorter oligomers of the invention which compriseother nucleotide analogues, such as high affinity nucleotide analogues.Indeed, the combination of LNA with other high affinity nucleotideanalogues are considered as part of the present invention.

Modification of nucleotides in positions 1 to 2, counting from the 3′end. The nucleotide at positions 1 and/or 2 may be a nucleotideanalogue, such as a high affinity nucleotide analogue, such as LNA, or anucleotide analogue selected from the group consisting of 2′-O-alkyl-RNAunit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, 2′-MOE-RNAunit, LNA unit, PNA unit, HNA unit, INA unit. The two 3′ nucleotide maytherefore be

Xx, xX, XX or xx, wherein: In one embodiment X is LNA and x is DNA oranother nucleotide analogue, such as a 2′ substituted nucleotideanalogue selected from the group consisting of 2′-O_alkyl-RNA unit,2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA, and a 2′MOERNA unit. Said non-LNA unit (x) may therefore be 2′MOE RNA or2′-fluoro-DNA. Alternatively X is a nucleotide analogue, and x is DNA.

The above modification at the 2 3′ terminal nucleotides may be combinedwith modification of nucleotides in positions 3-8 counting from the 3′end, as described below. In this respect nucleotides designated as X andx may be the same throughout the oligomer. It will be noted that whenthe oligomer is only 7 nucleotides in length the 8^(th) nucleotidecounting from the 3′ end should be discarded. In the followingembodiments which refer to the modification of nucleotides in positions3 to 8, counting from the 3′ end, the LNA units, in one embodiment, maybe replaced with other nucleotide analogues, such as those referred toherein. “X” may, therefore be selected from the group consisting of2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNAunit, 2′-MOE-RNA unit, LNA unit, PNA unit, HNA unit, INA unit. “x” ispreferably DNA or RNA, most preferably DNA. However, it is preferredthat X is LNA.

In one embodiment of the invention, the oligonucleotides of theinvention are modified in positions 3 to 8, counting from the 3′ end.The design of this sequence may be defined by the number of non-LNAunits present or by the number of LNA units present. In a preferredembodiment of the former, at least one, such as one, of the nucleotidesin positions three to eight, counting from the 3′ end, is a non-LNAunit. In another embodiment, at least two, such as two, of thenucleotides in positions three to eight, counting from the 3′ end, arenon-LNA units. In yet another embodiment, at least three, such as three,of the nucleotides in positions three to eight, counting from the 3′end, are non-LNA units. In still another embodiment, at least four, suchas four, of the nucleotides in positions three to eight, counting fromthe 3′ end, are non-LNA units. In a further embodiment, at least five,such as five, of the nucleotides in positions three to eight, countingfrom the 3′ end, are non-LNA units. In yet a further embodiment, all sixnucleotides in positions three to eight, counting from the 3′ end, arenon-LNA units.

Alternatively defined, in an embodiment, the oligonucleotide accordingto the present invention comprises at least three LNA units in positionsthree to eight, counting from the 3′ end. In an embodiment thereof, theoligonucleotide according to the present invention comprises three LNAunits in positions three to eight, counting from the 3′ end. Thesubstitution pattern for the nucleotides in positions three to eight,counting from the 3′ end, may be selected from the group consisting ofXXXxxx, xXXXxx, xxXXXx, xxxXXX, XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX,xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX andXxXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.In a preferred embodiment, the substitution pattern for the nucleotidesin positions three to eight, counting from the 3′ end, is selected fromthe group consisting of XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX,XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx,wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit. In amore preferred embodiment, the substitution pattern for the nucleotidesin positions three to eight, counting from the 3′ end, is selected fromthe group consisting of xXXxXx, xXXxxX, xxXXxX, xXxXXx, xXxxXX, xxXxXXand xXxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNAunit. In an embodiment, the substitution pattern for the nucleotides inpositions three to eight, counting from the 3′ end, is xXxXxX or XxXxXx,wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit. In anembodiment, the substitution pattern for the nucleotides in positionsthree to eight, counting from the 3′ end, is xXxXxX, wherein “X” denotesan LNA unit and “x” denotes a non-LNA unit.

In a further embodiment, the oligonucleotide according to the presentinvention comprises at least four LNA units in positions three to eight,counting from the 3′ end. In an embodiment thereof, the oligonucleotideaccording to the present invention comprises four LNA units in positionsthree to eight, counting from the 3′ end. The substitution pattern forthe nucleotides in positions three to eight, counting from the 3′ end,may be selected from the group consisting of xxXXXX, xXxXXX, xXXxXX,xXXXxX, xXXXXx, XXXX, XxXxXX, XxXXxX, XxXXXx, XXxxXX, XXxXxX, XXxXXx,XXXxxX, XXXxXx and XXXXxx, wherein “X” denotes an LNA unit and “x”denotes a non-LNA unit.

In yet a further embodiment, the oligonucleotide according to thepresent invention comprises at least five LNA units in positions threeto eight, counting from the 3′ end. In an embodiment thereof, theoligonucleotide according to the present invention comprises five LNAunits in positions three to eight, counting from the 3′ end. Thesubstitution pattern for the nucleotides in positions three to eight,counting from the 3′ end, may be selected from the group consisting ofxXXXXX, XxXXXX, XXxXXX, XXXxXX, XXXXxX and XXXXXx, wherein “X” denotesan LNA unit and “x” denotes a non-LNA unit.

Preferably, the oligonucleotide according to the present inventioncomprises one or two LNA units in positions three to eight, countingfrom the 3′ end. This is considered advantageous for the stability ofthe A-helix formed by the oligo:microRNA duplex, a duplex resembling anRNA:RNA duplex in structure.

In yet a further embodiment, the oligonucleotide according to thepresent invention comprises at least six LNA units in positions three toeight, counting from the 3′ end. In an embodiment thereof, theoligonucleotide according to the present invention comprises at fromthree to six LNA units in positions three to eight, counting from the 3′end, and in addition from none to three other high affinity nucleotideanalogues in the same region, such that the total amount of highaffinity nucleotide analogues (including the LNA units) amount to six inthe region from positions three to eight, counting from the 3′ end.

In some embodiments, such as when X is LNA, said non-LNA unit (x) isanother nucleotide analogue unit, such as a 2′ substituted nucleotideanalogue selected from the group consisting of 2′-O_alkyl-RNA unit,2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA, and a 2′MOERNA unit. Said non-LNA unit (x) may therefore be 2′MOE RNA or2′-fluoro-DNA.

For oligomers which have 9 or 10 nucleotides, the nucleotide atpositions 9 and/or 10 may be a nucleotide analogue, such as a highaffinity nucleotide analogue, such as LNA, or a nucleotide analogueselected from the group consisting of 2′-O-alkyl-RNA unit, 2′-OMe-RNAunit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, 2′-MOE-RNA unit, LNA unit,PNA unit, HNA unit, INA unit. The two 5′ nucleotides may therefore be

Xx, xX, XX or xx, wherein: In one embodiment X is LNA and x is DNA oranother nucleotide analogue, such as a 2′ substituted nucleotideanalogue selected from the group consisting of 2′-O_alkyl-RNA unit,2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA, and a 2′MOERNA unit. Said non-LNA unit (x) may therefore be 2′MOE RNA or2′-fluoro-DNA. Alternatively X is a nucleotide analogue, and x is DNA.

The above modification at the 2 5′ terminal nucleotides may be combinedwith modification of nucleotides in positions 3-8 counting from the 3′end, and/or the 2 3′ nucleotides as described above. In this respectnucleotides designated as X and x may be the same throughout theoligomer.

In a preferred embodiment of the invention, the oligonucleotideaccording to the present invention contains an LNA unit at the 5′ end.In another preferred embodiment, the oligonucleotide according to thepresent invention contains an LNA unit at the first two positions,counting from the 5′ end.

In one embodiment, the invention further provides for an oligomer asdescribed in the context of the pharmaceutical composition of theinvention, or for use in vivo in an organism, such as a medicament,wherein said oligomer (or contiguous nucleotide sequence) compriseseither

i) at least one phosphorothioate linkage and/or

ii) at least one 3′ terminal LNA unit, and/or

iii) at least one 5′ terminal LNA unit.

The oligomer may therefore contain at least one phosphorothioatelinkage, such as all linkages being phosphorothioates, and at least one3′ terminal LNA unit, and at least one 5′ terminal LNA unit.

It is preferable for most therapeutic uses that the oligonucleotide isfully phosphorothiolated—an exception being for therapeuticoligonucleotides for use in the CNS, such as in the brain or spine wherephosphorothioation can be toxic, and due to the absence of nucleases,phosphodiester bonds may be used, even between consecutive DNA units.

As referred to herein, other in one aspect of the oligonucleotideaccording to the invention is that the second 3′ nucleotide, and/or the9^(th) and 10^(th) (from the 3′ end), if present, may also be LNA.

In one embodiment, the oligomer comprises at least five nucleotideanalogue units, such as at least five LNA units, in positions which arecomplementary to the miRNA seed region.

In one embodiment, the nucleotide sequence of the oligomer which iscomplementary to the sequence of the microRNA seed region, is selectedfrom the group consisting of (X)xXXXXX, (X)XxXXXX, (X)XXxXXX, (X)XXXxXX,(X)XXXXxX and (X)XXXXXx, wherein “X” denotes a nucleotide analogue, suchas an LNA unit, (X) denotes an optional nucleotide analogue, such as anLNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the oligomer comprises six or seven nucleotideanalogue units, such as six or seven LNA units, in positions which arecomplementary to the miRNA seed region.

In one embodiment, the nucleotide sequence of the oligomer which iscomplementary to the sequence of the microRNA seed region, is selectedfrom the group consisting of XXXXXX, XxXXXXX, XXxXXXX, XXXxXXX, XXXXxXX,XXXXXxX and XXXXXXx, wherein “X” denotes a nucleotide analogue, such asan LNA unit, such as an LNA unit, and “x” denotes a DNA or RNAnucleotide unit.

In one embodiment, the two nucleotide motif at position 7 to 8, countingfrom the 3′ end of the oligomer is selected from the group consisting ofxx. XX, xX and Xx, wherein “X” denotes a nucleotide analogue, such as anLNA unit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotideunit.

In one embodiment, the two nucleotide motif at position 7 to 8, countingfrom the 3′ end of the oligomer is selected from the group consisting ofXX, xX and Xx, wherein “X” denotes a nucleotide analogue, such as an LNAunit, such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the oligomer comprises at 12 nucleotides and whereinthe two nucleotide motif at position 11 to 12, counting from the 3′ endof the oligomer is selected from the group consisting of xx, XX, xX andXx, wherein “X” denotes a nucleotide analogue, such as an LNA unit, suchas an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the oligomer comprises 12 nucleotides and wherein thetwo nucleotide motif at position 11 to 12, counting from the 3′ end ofthe oligomer is selected from the group consisting of XX, xX and Xx,wherein “X” denotes a nucleotide analogue, such as an LNA unit, such asan LNA unit, and “x” denotes a DNA or RNA nucleotide unit, such as a DNAunit.

In one embodiment, the oligomer comprises a nucleotide analogue unit,such as an LNA unit, at the 5′ end.

In one embodiment, the nucleotide analogue units, such as X, areindependently selected form the group consisting of: 2′-O-alkyl-RNAunit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, 2′-MOE-RNAunit, LNA unit, PNA unit, HNA unit, INA unit.

In one embodiment, all the nucleotides of the oligomer of the inventionare nucleotide analogue units.

In one embodiment, the nucleotide analogue units, such as X, areindependently selected form the group consisting of: 2′-OMe-RNA units,2′-fluoro-DNA units, and LNA units,

In one embodiment, the oligomer comprises said at least one LNA analogueunit and at least one further nucleotide analogue unit other than LNA.

In one embodiment, the non-LNA nucleotide analogue unit or units areindependently selected from 2′-OMe RNA units and 2′-fluoro DNA units.

In one embodiment, the oligomer consists of at least one sequence XYX orYXY, wherein X is LNA and Y is either a 2′-OMe RNA unit and 2′-fluoroDNA unit.

In one embodiment, the sequence of nucleotides of the oligomer consistsof alternative X and Y units.

In one embodiment, the oligomer comprises alternating LNA and DNA units(Xx) or (xX). In one embodiment, the oligomer comprises a motif ofalternating LNA followed by 2 DNA units (Xxx), xXx or xxX.

In one embodiment, at least one of the DNA or non-LNA nucleotideanalogue units are replaced with a LNA nucleotide in a position selectedfrom the positions identified as LNA nucleotide units in any one of theembodiments referred to above. In one embodiment, “X” donates an LNAunit.

Further Designs for Oligomers of the Invention

Table 1 below provides non-limiting examples of short microRNA sequencesthat could advantageously be targeted with an oligonucleotide of thepresent invention.

The oligonucleotides according to the invention, such as those disclosedin table 1 may, in one embodiment, have a sequence of 7, 8, 9 or 10 LNAnucleotides 5′-3′ LLLLLLL(L)(L)(L)(L), or have a sequence of nucleotidesselected form the group consisting of, the first 7, 8, 9 or 10nucleotides of the following motifs:

LdLddL(LXdXdXLXdXLXdXLXL), LdLdLL(LXdXdXLXLXLXdXLXL),LMLMML(L)(M)(M)(L)(M)(L)(M)(L)(L), LMLMLL(L)(M)(M)(L)(L)(L)(M)(L)(L),LFLFFL(L)(F)(F)(L)(F)(L)(F)(L)(L), LFLFLL(L)(F)(F)(L)(L)(L)(F)(L)(L),and every third designs such as; LddLdd(L)(d)(d)(L)(d)(d)(L)(d)(d)(L)(d)‘dLddLd(d)(L)(d)(d)(L)(d)(d)(L)(d)(d)(L),ddLddL(d)(d)(L)(d)(d)(L)(d)(d)(L)(d)(d),LMMLMM(L)(M)(M)(L)(M)(M)(L)(M)(M)(L)(M),MLMMLM(M)(L)(M)(M)(L)(M)(M)(L)(M)(M)(L),MMLMML(M)(M)(L)(M)(M)(L)(M)(M)(L)(M)(M),LFFLFF(L)(F)(F)(L)(F)(F)(L)(F)(F)(L)(F),FLFFLF(F)(L)(F)(F)(L)(F)(F)(L)(F)(F)(L),FFLFFL(F)(F)(L)(F)(F)(L)(F)(F)(L)(F)(F), anddLdLdL(d)(L)(d)(L)(d)(L)(d)(L)(d)(L)(d) and an every second design, suchas; LdLdLd(L)(d)(L)(d)(L)(d)(L)(d)(L)(d)(L),MLMLML(M)(L)(M)(L)(M)(L)(M)(L)(M)(L)(M),LMLMLM(L)(M)(L)(M)(L)(M)(L)(M)(L)(M)(L).FLFLFL(F)(L)(F)(L)(F)(L)(F)(L)(F)(L)(F), andLFLFLF(L)(F)(L)(F)(L)(F)(L)(F)(L)(F)(L); wherein L=LNA unit, d=DNAunits, M=2′MOE RNA. F=2′Fluoro and residues in brackets are optional.

Pharmaceutical Composition and Medical Application

The invention provides for a pharmaceutical composition comprising theoligomer according to the invention, and a pharmaceutically acceptablediluent, carrier, salt or adjuvant.

The invention further provides for the use of an oligonucleotideaccording to the invention, such as those which may form part of thepharmaceutical composition, for the manufacture of a medicament for thetreatment of a disease or medical disorder associated with the presenceor over-expression (upregulation) of the microRNA.

The invention further provides for a method for the treatment of adisease or medical disorder associated with the presence orover-expression of the microRNA, comprising the step of administering acomposition (such as the pharmaceutical composition) according to theinvention to a person in need of treatment.

The invention further provides for a method for reducing the effectiveamount of a miRNA in a cell or an organism, comprising administering acomposition (such as the pharmaceutical composition) according to theinvention or a oligomer according to the invention to the cell or theorganism. Reducing the effective amount in this context refers to thereduction of functional miRNA present in the cell or organism. It isrecognised that the preferred oligonucleotides according to theinvention may not always significantly reduce the actual amount of miRNAin the cell or organism as they typically form very stable duplexes withtheir miRNA targets. The reduction of the effective amount of the miRNAin a cell may, in one embodiment, be measured by detecting the level ofde-repression of the miRNA's target in the cell.

The invention further provides for a method for de-repression of atarget mRNA of a miRNA in a cell or an organism, comprisingadministering a composition (such as the pharmaceutical composition) ora oligomer according to the invention to the cell or the organism.

The invention further provides for the use of a oligomer of between 7-10such as 7, 8, 9, or 10 nucleotides in length, for the manufacture of amedicament for the treatment of a disease or medical disorder associatedwith the presence or over-expression of the microRNA.

In one embodiment the medical condition (or disease) is hepatitis C(HCV), and the miRNA is miR-122.

In one embodiment, the pharmaceutical composition according to theinvention is for use in the treatment of a medical disorder or diseaseselected from the group consisting of: hepatitis C virus infection andhypercholesterolemia and related disorders, and cancers.

In one embodiment the medical disorder or disease is a CNS disease, suchas a CNS disease where one or more microRNAs are known to be indicated.

In the context of hypercholesterolemia related disorders refers todiseases such as atherosclerosis or hyperlipidemia. Further examples ofrelated diseases also include different types of HDL/LDL cholesterolimbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL),acquired hyperlipidemia, statin-resistant hypercholesterolemia; coronaryartery disease (CAD) coronary heart disease (CHD), atherosclerosis.

In one embodiment, the pharmaceutical composition according to theinvention further comprises a second independent active ingredient thatis an inhibitor of the VLDL assembly pathway, such as an ApoB inhibitor,or an MTP inhibitor (such as those disclosed in U.S. 60/977,497, herebyincorporated by reference).

The invention further provides for a method for the treatment of adisease or medical disorder associated with the presence orover-expression of the microRNA, comprising the step of administering acomposition (such as the pharmaceutical composition) comprising aoligomer of between 7-10 such as 7, 8, 9, or 10 nucleotides in length,to a person in need of treatment.

The invention further provides for a method for reducing the effectiveamount of a miRNA target (i.e. ‘available’ miRNA) in a cell or anorganism, comprising administering a composition (such as thepharmaceutical composition) comprising a oligomer of between 6 7-10 suchas 7, 8, 9, or 10 nucleotides in length, to the cell or the organism.

It should be recognised that “reducing the effective amount” of one ormore microRNAs in a cell or organism, refers to the inhibition of themicroRNA function in the call or organism. The cell is preferably amammalian cell or a human cell which expresses the microRNA ormicroRNAs.

The invention further provides for a method for de-repression of atarget mRNA of a miRNA in a cell or an organism, comprising a oligomerof 7-10 such as 7, 8, 9, or 10 nucleotides in length, or (or acomposition comprising said oligonucleotide) to the cell or theorganism.

As mentioned above, microRNAs are related to a number of diseases.Hence, a fourth aspect of the invention relates to the use of anoligonucleotide as defined herein for the manufacture of a medicamentfor the treatment of a disease associated with the expression ofmicroRNAs selected from the group consisting of spinal muscular atrophy,Tourette's syndrome, hepatitis C, fragile X mental retardation, DiGeorgesyndrome and cancer, such as in non limiting example, chroniclymphocytic leukemia, breast cancer, lung cancer and colon cancer, inparticular cancer.

Methods of Synthesis

The invention further provides for a method for the synthesis of anoligomer targeted against a human microRNA, such as an oligomerdescribed herein, said method comprising the steps of:

-   a. Optionally selecting a first nucleotide, counting from the 3′    end, which is a nucleotide analogue, such as an LNA nucleotide.-   b. Optionally selecting a second nucleotide, counting from the 3′    end, which is a nucleotide analogue, such as an LNA nucleotide.-   c. Selecting a region of the oligomer which corresponds to the miRNA    seed region, wherein said region is as defined herein.-   d. Selecting a seventh and optionally an eight nucleotides defined    herein.-   e. Optionally selecting one or two further 5′ terminal of the    oligomer is as defined herein;

wherein the synthesis is performed by sequential synthesis of theregions defined in steps a-e, wherein said synthesis may be performed ineither the 3′-5′ (a to f) or 5′-3′ (e to a) direction, and wherein saidoligomer is complementary to a sequence of the miRNA target.

The invention further provides for a method for the preparation of anoligomer (such as an oligomer according to the invention), said methodcomprising the steps of a) comparing the sequences of two or more miRNAsequences to identify two or more miRNA sequences which comprise acommon contiguous nucleotide sequence of at least 7 nucleotides inlength, such as 7, 8, 9 or 10 nucleotides in length (i.e. a sequencefound in both non-identical miRNAs), b) preparing an oligomer sequencewhich consists or comprises of a contiguous nucleotide sequence with iscomplementary to said common contiguous nucleotide sequence, whereinsaid oligomer is, as according to the oligomer of the invention. In apreferred example, the common contiguous nucleotide sequence consists orcomprises of the seed region of each of said two or more miRNA sequences(which comprise a common contiguous nucleotide sequence of at least 6nucleotides in length). In one embodiment, the seed regions of the twoor more miRNAs are identical. Suitably the oligomer consists orcomprises a seedmer sequence of 7 or 8 nucleotides in length whichcomprises of a sequence which is complementary to said two or moremiRNAs. This method may be used in conjunction with step c of the abovemethod.

The method for the synthesis of the oligomer according to the inventionmay be performed using standard solid phase oligonucleotide synthesis.

In one embodiment, the method for the synthesis of a oligomer targetedagainst a human microRNA, is performed in the 3′ to 5′ direction a-e.

A further aspect of the invention is a method to reduce the levels oftarget microRNA by contacting the target microRNA to an oligonucleotideas defined herein, wherein the oligonucleotide (i) is complementary tothe target microRNA sequence (ii) does not contain a nucleotide at the3′ end that corresponds to the first 5′ end nucleotide of the targetmicroRNA.

Duplex Stability and T_(m)

In one embodiment, the oligomer of the invention is capable of forming aduplex with a complementary single stranded RNA nucleic acid molecule(typically of about the same length of said single strandedoligonucleotide) with phosphodiester internucleoside linkages, whereinthe duplex has a T_(m) of between 30° C. and 70° C. or 80° C., such asbetween 30° C. and 60° C. to 70° C., or between 30° C. and 50° C. or 60°C. In one embodiment the T_(m) is at least 40° C. T_(m) may bedetermined by determining the T_(m) of the oligomer and a complementaryRNA target in the following buffer conditions: 100 mM NaCl, 0.1 mM EDTA,10 mM Na-phosphate, pH 7.0 (see examples for a detailed protocol). Ahigh affinity analogue may be defined as an analogue which, when used inthe oligomer of the invention, results in an increase in the T_(m) ofthe oligomer as compared to an identical oligomer which has containsonly DNA bases.

Conjugates

In one embodiment, said oligomer is conjugated with one or morenon-nucleotide (or polynucleotide) compounds.

In the context the term “conjugate” is intended to indicate aheterogenous molecule formed by the covalent attachment (“conjugation”)of the oligomer as described herein to one or more non-nucleotide, ornon-polynucleotide moieties. Examples of non-nucleotide ornon-polynucleotide moieties include macromolecular agents such asproteins, fatty acid chains, sugar residues, glycoproteins, polymers, orcombinations thereof. Typically proteins may be antibodies for a targetprotein. Typical polymers may be polyethylene glycol.

Therefore, in various embodiments, the oligomer of the invention maycomprise both a polynucleotide region which typically consists of acontiguous sequence of nucleotides, and a further non-nucleotide region.When referring to the oligomer of the invention consisting of acontiguous nucleotide sequence, the compound may comprise non-nucleotidecomponents, such as a conjugate component.

In various embodiments of the invention the oligomeric compound islinked to ligands/conjugates, which may be used, e.g. to increase thecellular uptake of oligomeric compounds. WO2007/031091 provides suitableligands and conjugates, which are hereby incorporated by reference.

The invention also provides for a conjugate comprising the compoundaccording to the invention as herein described, and at least onenon-nucleotide or non-polynucleotide moiety covalently attached to saidcompound. Therefore, in various embodiments where the compound of theinvention consists of a specified nucleic acid or nucleotide sequence,as herein disclosed, the compound may also comprise at least onenon-nucleotide or non-polynucleotide moiety (e.g. not comprising one ormore nucleotides or nucleotide analogues) covalently attached to saidcompound.

Conjugation (to a conjugate moiety) may enhance the activity, cellulardistribution or cellular uptake of the oligomer of the invention. Suchmoieties include, but are not limited to, antibodies, polypeptides,lipid moieties such as a cholesterol moiety, cholic acid, a thioether,e.g. Hexyl-s-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipids, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-o-hexadecyl-rac-glycero-3-h-phosphonate, a polyamine or apolyethylene glycol chain, an adamantane acetic acid, a palmityl moiety,an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

The oligomers of the invention may also be conjugated to active drugsubstances, for example, aspirin, ibuprofen, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic.

In certain embodiments the conjugated moiety is a sterol, such ascholesterol.

In various embodiments, the conjugated moiety comprises or consists of apositively charged polymer, such as a positively charged peptides of,for example between 1-50, such as 2-20 such as 3-10 amino acid residuesin length, and/or polyalkylene oxide such as polyethylglycol(PEG) orpolypropylene glycol—see WO 2008/034123, hereby incorporated byreference. Suitably the positively charged polymer, such as apolyalkylene oxide may be attached to the oligomer of the invention viaa linker such as the releasable linker described in WO 2008/034123.

By way of example, the following conjugate moieties may be used in theconjugates of the invention:

Activated Oligomers

The term “activated oligomer,” as used herein, refers to an oligomer ofthe invention that is covalently linked (i.e., functionalized) to atleast one functional moiety that permits covalent linkage of theoligomer to one or more conjugated moieties, i.e., moieties that are notthemselves nucleic acids or monomers, to form the conjugates hereindescribed. Typically, a functional moiety will comprise a chemical groupthat is capable of covalently bonding to the oligomer via, e.g., a3′-hydroxyl group or the exocyclic NH₂ group of the adenine base, aspacer that is preferably hydrophilic and a terminal group that iscapable of binding to a conjugated moiety (e.g., an amino, sulfhydryl orhydroxyl group). In some embodiments, this terminal group is notprotected. e.g., is an NH₂ group. In other embodiments, the terminalgroup is protected, for example, by any suitable protecting group suchas those described in “Protective Groups in Organic Synthesis” byTheodora W Greene and Peter G M Wuts, 3rd edition (John Wiley & Sons,1999). Examples of suitable hydroxyl protecting groups include esterssuch as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, ortriphenylmethyl, and tetrahydropyranyl. Examples of suitable aminoprotecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl,triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groupssuch as trichloroacetyl or trifluoroacetyl. In some embodiments, thefunctional moiety is self-cleaving. In other embodiments, the functionalmoiety is biodegradable. See e.g., U.S. Pat. No. 7,087,229, which isincorporated by reference herein in its entirety.

In some embodiments, oligomers of the invention are functionalized atthe 5′ end in order to allow covalent attachment of the conjugatedmoiety to the 5′ end of the oligomer. In other embodiments, oligomers ofthe invention can be functionalized at the 3′ end. In still otherembodiments, oligomers of the invention can be functionalized along thebackbone or on the heterocyclic base moiety. In yet other embodiments,oligomers of the invention can be functionalized at more than oneposition independently selected from the 5′ end, the 3′ end, thebackbone and the base.

In some embodiments, activated oligomers of the invention aresynthesized by incorporating during the synthesis one or more monomersthat is covalently attached to a functional moiety. In otherembodiments, activated oligomers of the invention are synthesized withmonomers that have not been functionalized, and the oligomer isfunctionalized upon completion of synthesis. In some embodiments, theoligomers are functionalized with a hindered ester containing anaminoalkyl linker, wherein the alkyl portion has the formula (CH₂)_(w),wherein w is an integer ranging from 1 to 10, preferably about 6,wherein the alkyl portion of the alkylamino group can be straight chainor branched chain, and wherein the functional group is attached to theoligomer via an ester group (—O—C(O)—(CH₂)_(w)NH).

In other embodiments, the oligomers are functionalized with a hinderedester containing a (CH₂)_(w)-sulfhydryl (SH) linker, wherein w is aninteger ranging from 1 to 10, preferably about 6, wherein the alkylportion of the alkylamino group can be straight chain or branched chain,and wherein the functional group attached to the oligomer via an estergroup (—O—C(O)—(CH₂)_(w)SH).

In some embodiments, sulfhydryl-activated oligonucleotides areconjugated with polymer moieties such as polyethylene glycol or peptides(via formation of a disulfide bond).

Activated oligomers containing hindered esters as described above can besynthesized by any method known in the art, and in particular by methodsdisclosed in PCT Publication No. WO 2008/034122 and the examplestherein, which is incorporated herein by reference in its entirety.

In still other embodiments, the oligomers of the invention arefunctionalized by introducing sulfhydryl, amino or hydroxyl groups intothe oligomer by means of a functionalizing reagent substantially asdescribed in U.S. Pat. Nos. 4,962,029 and 4,914,210, i.e., asubstantially linear reagent having a phosphoramidite at one end linkedthrough a hydrophilic spacer chain to the opposing end which comprises aprotected or unprotected sulfhydryl, amino or hydroxyl group. Suchreagents primarily react with hydroxyl groups of the oligomer. In someembodiments, such activated oligomers have a functionalizing reagentcoupled to a 5′-hydroxyl group of the oligomer. In other embodiments,the activated oligomers have a functionalizing reagent coupled to a3′-hydroxyl group. In still other embodiments, the activated oligomersof the invention have a functionalizing reagent coupled to a hydroxylgroup on the backbone of the oligomer. In yet further embodiments, theoligomer of the invention is functionalized with more than one of thefunctionalizing reagents as described in U.S. Pat. Nos. 4,962,029 and4,914,210, incorporated herein by reference in their entirety. Methodsof synthesizing such functionalizing reagents and incorporating theminto monomers or oligomers are disclosed in U.S. Pat. Nos. 4,962,029 and4,914,210.

In some embodiments, the 5′-terminus of a solid-phase bound oligomer isfunctionalized with a dienyl phosphoramidite derivative, followed byconjugation of the deprotected oligomer with, e.g., an amino acid orpeptide via a Diels-Alder cycloaddition reaction.

In various embodiments, the incorporation of monomers containing2′-sugar modifications, such as a 2′-carbamate substituted sugar or a2′-(O-pentyl-N-phthalimido)-deoxyribose sugar into the oligomerfacilitates covalent attachment of conjugated moieties to the sugars ofthe oligomer. In other embodiments, an oligomer with an amino-containinglinker at the 2-position of one or more monomers is prepared using areagent such as, for example,5′-dimethoxytrityl-2-O-(e-phthalimidylaminopentyl)-2′-deoxyadenosine-3′-N,N-diisopropyl-cyanoethoxyphosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters,1991, 34, 7171.

In still further embodiments, the oligomers of the invention may haveamine-containing functional moieties on the nucleotide, including on theN6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or5 positions of cytosine. In various embodiments, such functionalizationmay be achieved by using a commercial reagent that is alreadyfunctionalized in the oligomer synthesis.

Some functional moieties are commercially available, for example,heterobifunctional and homobifunctional linking moieties are availablefrom the Pierce Co. (Rockford, Ill.). Other commercially availablelinking groups are 5′-Amino-Modifier C6 and 3′-Amino-Modifier reagents,both available from Glen Research Corporation (Sterling, Va.).5′-Amino-Modifier C6 is also available from ABI (Applied BiosystemsInc., Foster City. Calif.) as Aminolink-2, and 3′-Amino-Modifier is alsoavailable from Clontech Laboratories Inc. (Palo Alto, Calif.).

Therapy and Pharmaceutical Compositions—Formulation and Administration

As explained initially, the oligonucleotides of the invention willconstitute suitable drugs with improved properties. The design of apotent and safe drug requires the fine-tuning of various parameters suchas affinity/specificity, stability in biological fluids, cellularuptake, mode of action, pharmacokinetic properties and toxicity.

Accordingly, in a further aspect the present invention relates to apharmaceutical composition comprising an oligonucleotide according tothe invention and a pharmaceutically acceptable diluent, carrier oradjuvant. Preferably said carrier is saline or buffered saline.

In a still further aspect the present invention relates to anoligonucleotide according to the present invention for use as amedicament.

As will be understood, dosing is dependent on severity andresponsiveness of the disease state to be treated, and the course oftreatment lasting from several days to several months, or until a cureis effected or a diminution of the disease state is achieved. Optimaldosing schedules can be calculated from measurements of drugaccumulation in the body of the patient. Optimum dosages may varydepending on the relative potency of individual oligonucleotides.Generally it can be estimated based on EC50s found to be effective in invitro and in vivo animal models. In general, dosage is from 0.01 μg to 1g per kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 10 years or by continuousinfusion for hours up to several months. The repetition rates for dosingcan be estimated based on measured residence times and concentrations ofthe drug in bodily fluids or tissues. Following successful treatment, itmay be desirable to have the patient undergo maintenance therapy toprevent the recurrence of the disease state.

As indicated above, the invention also relates to a pharmaceuticalcomposition, which comprises at least one oligonucleotide of theinvention as an active ingredient. It should be understood that thepharmaceutical composition according to the invention optionallycomprises a pharmaceutical carrier, and that the pharmaceuticalcomposition optionally comprises further compounds, such aschemotherapeutic compounds, anti-inflammatory compounds, antiviralcompounds and/or immuno-modulating compounds.

The oligonucleotides of the invention can be used “as is” or in form ofa variety of pharmaceutically acceptable salts. As used herein, the term“pharmaceutically acceptable salts” refers to salts that retain thedesired biological activity of the herein-identified oligonucleotidesand exhibit minimal undesired toxicological effects. Non-limitingexamples of such salts can be formed with organic amino acid and baseaddition salts formed with metal cations such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium,potassium, and the like, or with a cation formed from ammonia,N,N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, orethylenediamine.

In one embodiment of the invention, the oligonucleotide may be in theform of a pro-drug. Oligonucleotides are by virtue negatively chargedions. Due to the lipophilic nature of cell membranes the cellular uptakeof oligonucleotides are reduced compared to neutral or lipophilicequivalents. This polarity “hindrance” can be avoided by using thepro-drug approach (see e.g. Crooke, R. M. (1998) in Crooke, S. T.Antisense research and Application. Springer-Verlag, Berlin, Germany,vol. 131, pp. 103-140).

Pharmaceutically acceptable binding agents and adjuvants may comprisepart of the formulated drug.

Examples of delivery methods for delivery of the therapeutic agentsdescribed herein, as well as details of pharmaceutical formulations,salts, may are well described elsewhere for example in U.S. provisionalapplication 60/838,710 and 60/788,995, which are hereby incorporated byreference, and Danish applications, PA 2006 00615 which is also herebyincorporated by reference.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Delivery ofdrug to tumour tissue may be enhanced by carrier-mediated deliveryincluding, but not limited to, cationic liposomes, cyclodextrins,porphyrin derivatives, branched chain dendrimers, polyethyleniminepolymers, nanoparticles and microspheres (Dass C R. J Pharm Pharmacol2002; 54(1):3-27). The pharmaceutical formulations of the presentinvention, which may conveniently be presented in unit dosage form, maybe prepared according to conventional techniques well known in thepharmaceutical industry. Such techniques include the step of bringinginto association the active ingredients with the pharmaceuticalcarrier(s) or excipient(s). In general the formulations are prepared byuniformly and intimately bringing into association the activeingredients with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product. The compositions ofthe present invention may be formulated into any of many possible dosageforms such as, but not limited to, tablets, capsules, gel capsules,liquid syrups, soft gels and suppositories. The compositions of thepresent invention may also be formulated as suspensions in aqueous,non-aqueous or mixed media. Aqueous suspensions may further containsubstances which increase the viscosity of the suspension including, forexample, sodium carboxymethylcellulose, sorbitol and/or dextran. Thesuspension may also contain stabilizers. The compounds of the inventionmay also be conjugated to active drug substances, for example, aspirin,ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or anantibiotic.

In another embodiment, compositions of the invention may contain one ormore oligonucleotide compounds, targeted to a first microRNA and one ormore additional oligonucleotide compounds targeted to a second microRNAtarget. Two or more combined compounds may be used together orsequentially.

The compounds disclosed herein are useful for a number of therapeuticapplications as indicated above. In general, therapeutic methods of theinvention include administration of a therapeutically effective amountof an oligonucleotide to a mammal, particularly a human. In a certainembodiment, the present invention provides pharmaceutical compositionscontaining (a) one or more compounds of the invention, and (b) one ormore chemotherapeutic agents. When used with the compounds of theinvention, such chemotherapeutic agents may be used individually,sequentially, or in combination with one or more other suchchemotherapeutic agents or in combination with radiotherapy. Allchemotherapeutic agents known to a person skilled in the art are hereincorporated as combination treatments with compound according to theinvention. Other active agents, such as anti-inflammatory drugs,including but not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, antiviral drugs, and immuno-modulating drugs may alsobe combined in compositions of the invention. Two or more combinedcompounds may be used together or sequentially.

Examples of therapeutic indications which may be treated by thepharmaceutical compositions of the invention:

microRNA Possible medical indications miR-1 Cardiac arythmia miR-21Glioblastoma, breast cancer, hepatocellular carcinoma, colorectalcancer, sensitization of gliomas to cytotoxic drugs, cardiac hypertrophymiR-21, Response to chemotherapy and regulation of miR-200bcholangiocarcinoma growth and miR-141 miR-122 hypercholesterolemia,hepatitis C infection, hemochromatosis miR-19b lymphoma and other tumourtypes miR-26a Osteoblast differentiation of human stem cells miR-155lymphoma, pancreatic tumor development, breast and lung cancer miR-203Psoriasis miR-375 diabetes, metabolic disorders, glucose-induced insulinsecretion from pancreatic endocrine cells miR-181 myoblastdifferentiation, auto immune disorders miR-10b Breast cancer cellinvasion and metastasis miR-125b-1 Breast, lung, ovarian and cervicalcancer miR-221 Prostate carcinoma, human thyroid papillary car, humanand 222 hepatocellular carcinoma miRNA-372 testicular germ cell tumors.and - 373 miR-142 B-cell leukemia miR-17-19b B-cell lymphomas, lungcancer, hepatocellular carcinoma cluster

Tumor suppressor gene tropomysin 1 (TPM1) mRNA has been indicated as atarget of miR-21. Myotrophin (mtpn) mRNA has been indicated as a targetof miR 375.

In an even further aspect, the present invention relates to the use ofan oligonucleotide according to the invention for the manufacture of amedicament for the treatment of a disease selected from the groupconsisting of: atherosclerosis, hypercholesterolemia and hyperlipidemia;cancer, glioblastoma, breast cancer, lymphoma, lung cancer; diabetes,metabolic disorders; myoblast differentiation; immune disorders.

The invention further refers to oligonucleotides according to theinvention for the use in the treatment of from a disease selected fromthe group consisting of: atherosclerosis, hypercholesterolemia andhyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma, lungcancer; diabetes, metabolic disorders; myoblast differentiation; immunedisorders.

The invention provides for a method of treating a subject suffering froma disease or condition selected from the group consisting of:atherosclerosis, hypercholesterolemia and hyperlipidemia; cancer,glioblastoma, breast cancer, lymphoma, lung cancer; diabetes, metabolicdisorders; myoblast differentiation; immune disorders, the methodcomprising the step of administering an oligonucleotide orpharmaceutical composition of the invention to the subject in needthereof.

The invention further provides for a kit comprising a pharmaceuticalcomposition according to the invention, and a second independent activeingredient that is an inhibitor of the VLDL assembly pathway, such as anApoB inhibitor, or an MTP inhibitor.

Cancer

In an even further aspect, the present invention relates to the use ofan oligonucleotide according to the invention for the manufacture of amedicament for the treatment of cancer. In another aspect, the presentinvention concerns a method for treatment of, or prophylaxis against,cancer, said method comprising administering an oligonucleotide of theinvention or a pharmaceutical composition of the invention to a patientin need thereof.

Such cancers may include lymphoreticular neoplasia, lymphoblasticleukemia, brain tumors, gastric tumors, plasmacytomas, multiple myeloma,leukemia, connective tissue tumors, lymphomas, and solid tumors.

In the use of a compound of the invention for the manufacture of amedicament for the treatment of cancer, said cancer may suitably be inthe form of a solid tumor. Analogously, in the method for treatingcancer disclosed herein said cancer may suitably be in the form of asolid tumor.

Furthermore, said cancer is also suitably a carcinoma. The carcinoma istypically selected from the group consisting of malignant melanoma,basal cell carcinoma, ovarian carcinoma, breast carcinoma, non-smallcell lung cancer, renal cell carcinoma, bladder carcinoma, recurrentsuperficial bladder cancer, stomach carcinoma, prostatic carcinoma,pancreatic carcinoma, lung carcinoma, cervical carcinoma, cervicaldysplasia, laryngeal papillomatosis, colon carcinoma, colorectalcarcinoma and carcinoid tumors. More typically, said carcinoma isselected from the group consisting of malignant melanoma, non-small celllung cancer, breast carcinoma, colon carcinoma and renal cell carcinoma.The malignant melanoma is typically selected from the group consistingof superficial spreading melanoma, nodular melanoma, lentigo malignamelanoma, acral melagnoma, amelanotic melanoma and desmoplasticmelanoma.

Alternatively, the cancer may suitably be a sarcoma. The sarcoma istypically in the form selected from the group consisting ofosteosarcoma. Ewing's sarcoma, chondrosarcoma, malignant fibroushistiocytoma, fibrosarcoma and Kaposi's sarcoma.

Alternatively, the cancer may suitably be a glioma.

A further embodiment is directed to the use of an oligonucleotideaccording to the invention for the manufacture of a medicament for thetreatment of cancer, wherein said medicament further comprises achemotherapeutic agent selected from the group consisting ofadrenocorticosteroids, such as prednisone, dexamethasone or decadron;altretamine (hexalen, hexamethylmelamine (HMM)); amifostine (ethyol);aminoglutethimide (cytadren); amsacrine (M-AMSA); anastrozole(arimidex); androgens, such as testosterone; asparaginase (elspar);bacillus calmette-gurin; bicalutamide (casodex); bleomycin (blenoxane);busulfan (myleran); carboplatin (paraplatin); carmustine (BCNU, BiCNU);chlorambucil (leukeran); chlorodeoxyadenosine (2-CDA, cladribine,leustatin); cisplatin (platinol); cytosine arabinoside (cytarabine);dacarbazine (DTIC); dactinomycin (actinomycin-D, cosmegen); daunorubicin(cerubidine); docetaxel (taxotere); doxorubicin (adriomycin);epirubicin; estramustine (emcyt); estrogens, such as diethylstilbestrol(DES); etopside (VP-16, VePesid, etopophos); fludarabine (fludara);flutamide (eulexin); 5-FUDR (floxuridine); 5-fluorouracil (5-FU);gemcitabine (gemzar); goserelin (zodalex); herceptin (trastuzumab);hydroxyurea (hydrea); idarubicin (idamycin); ifosfamide; IL-2(proleukin, aldesleukin); interferon alpha (intron A, roferon A);irinotecan (camptosar); leuprolide (lupron); levamisole (ergamisole);lomustine (CCNU); mechlorathamine (mustargen, nitrogen mustard);melphalan (alkeran); mercaptopurine (purinethol, 6-MP); methotrexate(mexate); mitomycin-C(mutamucin); mitoxantrone (novantrone); octreotide(sandostatin); pentostatin (2-deoxycoformycin, nipent); plicamycin(mithramycin, mithracin); prorocarbazine (matulane); streptozocin;tamoxifin (nolvadex); taxol (paclitaxel); teniposide (vumon, VM-26);thiotepa; topotecan (hycamtin); tretinoin (vesanoid, all-trans retinoicacid); vinblastine (valban); vincristine (oncovin) and vinorelbine(navelbine). Suitably, the further chemotherapeutic agent is selectedfrom taxanes such as Taxol, Paclitaxel or Docetaxel.

Similarly, the invention is further directed to the use of anoligonucleotide according to the invention for the manufacture of amedicament for the treatment of cancer, wherein said treatment furthercomprises the administration of a further chemotherapeutic agentselected from the group consisting of adrenocorticosteroids, such asprednisone, dexamethasone or decadron; altretamine (hexalen,hexamethylmelamine (HMM)); amifostine (ethyol); aminoglutethimide(cytadren); amsacrine (M-AMSA); anastrozole (arimidex); androgens, suchas testosterone; asparaginase (elspar); bacillus calmette-gurin;bicalutamide (casodex); bleomycin (blenoxane); busulfan (myleran);carboplatin (paraplatin); carmustine (BCNU, BiCNU); chlorambucil(leukeran); chlorodeoxyadenosine (2-CDA, cladribine, leustatin);cisplatin (platinol); cytosine arabinoside (cytarabine); dacarbazine(DTIC); dactinomycin (actinomycin-D, cosmegen); daunorubicin(cerubidine); docetaxel (taxotere); doxorubicin (adriomycin);epirubicin; estramustine (emcyt); estrogens, such as diethylstilbestrol(DES); etopside (VP-16, VePesid, etopophos); fludarabine (fludara);flutamide (eulexin); 5-FUDR (floxuridine); 5-fluorouracil (5-FU);gemcitabine (gemzar); goserelin (zodalex); herceptin (trastuzumab);hydroxyurea (hydrea); idarubicin (idamycin); ifosfamide; IL-2(proleukin, aldesleukin); interferon alpha (intron A, roferon A);irinotecan (camptosar); leuprolide (lupron); levamisole (ergamisole);lomustine (CCNU); mechlorathamine (mustargen, nitrogen mustard);melphalan (alkeran); mercaptopurine (purinethol, 6-MP); methotrexate(mexate); mitomycin-C(mutamucin); mitoxantrone (novantrone); octreotide(sandostatin); pentostatin (2-deoxycoformycin, nipent); plicamycin(mithramycin, mithracin); prorocarbazine (matulane); streptozocin;tamoxifin (nolvadex); taxol (paclitaxel); teniposide (vumon, VM-26);thiotepa; topotecan (hycamtin); tretinoin (vesanoid, all-trans retinoicacid); vinblastine (valban); vincristine (oncovin) and vinorelbine(navelbine). Suitably, said treatment further comprises theadministration of a further chemotherapeutic agent selected fromtaxanes, such as Taxol, Paclitaxel or Docetaxel.

Alternatively stated, the invention is furthermore directed to a methodfor treating cancer, said method comprising administering anoligonucleotide of the invention or a pharmaceutical compositionaccording to the invention to a patient in need thereof and furthercomprising the administration of a further chemotherapeutic agent. Saidfurther administration may be such that the further chemotherapeuticagent is conjugated to the compound of the invention, is present in thepharmaceutical composition, or is administered in a separateformulation.

Infectious Diseases

It is contemplated that the compounds of the invention may be broadlyapplicable to a broad range of infectious diseases, such as diphtheria,tetanus, pertussis, polio, hepatitis B, hepatitis C, hemophilusinfluenza, measles, mumps, and rubella.

Hsa-miR122 (SEQ ID NO: 150) is indicated in hepatitis C infection and assuch oligonucleotides according to the invention which target miR-122may be used to treat Hepatitis C infection.

Accordingly, in yet another aspect the present invention relates the useof an oligonucleotide according to the invention for the manufacture ofa medicament for the treatment of an infectious disease, as well as to amethod for treating an infectious disease, said method comprisingadministering an oligonucleotide according to the invention or apharmaceutical composition according to the invention to a patient inneed thereof.

In a preferred embodiment, the invention provides for a combinationtreatment providing an anti miR-122 oligomer in combination with aninhibitor of VLDL assembly, such as an inhibitor of apoB, or of MTP.

Inflammatory Diseases

The inflammatory response is an essential mechanism of defense of theorganism against the attack of infectious agents, and it is alsoimplicated in the pathogenesis of many acute and chronic diseases,including autoimmune disorders. In spite of being needed to fightpathogens, the effects of an inflammatory burst can be devastating. Itis therefore often necessary to restrict the symptomatology ofinflammation with the use of anti-inflammatory drugs. Inflammation is acomplex process normally triggered by tissue injury that includesactivation of a large array of enzymes, the increase in vascularpermeability and extravasation of blood fluids, cell migration andrelease of chemical mediators, all aimed to both destroy and repair theinjured tissue.

In yet another aspect, the present invention relates to the use of anoligonucleotide according to the invention for the manufacture of amedicament for the treatment of an inflammatory disease, as well as to amethod for treating an inflammatory disease, said method comprisingadministering an oligonucleotide according to the invention or apharmaceutical composition according to the invention to a patient inneed thereof.

In one preferred embodiment of the invention, the inflammatory diseaseis a rheumatic disease and/or a connective tissue diseases, such asrheumatoid arthritis, systemic lupus erythematous (SLE) or Lupus,scleroderma, polymyositis, inflammatory bowel disease, dermatomyositis,ulcerative colitis, Crohn's disease, vasculitis, psoriatic arthritis,exfoliative psoriatic dermatitis, pemphigus vulgaris and Sjorgren'ssyndrome, in particular inflammatory bowel disease and Crohn's disease.

Alternatively, the inflammatory disease may be a non-rheumaticinflammation, like bursitis, synovitis, capsulitis, tendinitis and/orother inflammatory lesions of traumatic and/or sportive origin.

Metabolic Diseases

A metabolic disease is a disorder caused by the accumulation ofchemicals produced naturally in the body. These diseases are usuallyserious, some even life threatening. Others may slow physicaldevelopment or cause mental retardation. Most infants with thesedisorders, at first, show no obvious signs of disease. Proper screeningat birth can often discover these problems. With early diagnosis andtreatment, metabolic diseases can often be managed effectively.

In yet another aspect, the present invention relates to the use of anoligonucleotide according to the invention or a conjugate thereof forthe manufacture of a medicament for the treatment of a metabolicdisease, as well as to a method for treating a metabolic disease, saidmethod comprising administering an oligonucleotide according to theinvention or a conjugate thereof, or a pharmaceutical compositionaccording to the invention to a patient in need thereof.

In one preferred embodiment of the invention, the metabolic disease isselected from the group consisting of Amyloidosis. Biotinidase, OMIM(Online Mendelian Inheritance in Man), Crigler Najjar Syndrome,Diabetes, Fabry Support & Information Group, Fatty acid OxidationDisorders, Galactosemia, Glucose-6-Phosphate Dehydrogenase (G6PD)deficiency, Glutaric aciduria, International Organization of GlutaricAcidemia, Glutaric Acidemia Type I, Glutaric Acidemia, Type II, GlutaricAcidemia Type I, Glutaric Acidemia Type-II, F-HYPDRR—FamilialHypophosphatemia, Vitamin D Resistant Rickets, Krabbe Disease, Longchain 3 hydroxyacyl CoA dehydrogenase deficiency (LCHAD), MannosidosisGroup, Maple Syrup Urine Disease, Mitochondrial disorders,Mucopolysaccharidosis Syndromes: Niemann Pick, Organic acidemias, PKU,Pompe disease, Porphyria, Metabolic Syndrome, Hyperlipidemia andinherited lipid disorders, Trimethylaminuria: the fish malodor syndrome,and Urea cycle disorders.

Liver Disorders

In yet another aspect, the present invention relates to the use of anoligonucleotide according to the invention or a conjugate thereof forthe manufacture of a medicament for the treatment of a liver disorder,as well as to a method for treating a liver disorder, said methodcomprising administering an oligonucleotide according to the inventionor a conjugate thereof, or a pharmaceutical composition according to theinvention to a patient in need thereof.

In one preferred embodiment of the invention, the liver disorder isselected from the group consisting of Biliary Atresia, AlagilleSyndrome, Alpha-1 Antitrypsin, Tyrosinemia, Neonatal Hepatitis, andWilson Disease.

Other Uses

The oligonucleotides of the present invention can be utilized for asresearch reagents for diagnostics, therapeutics and prophylaxis. Inresearch, the oligonucleotide may be used to specifically inhibit thesynthesis of target genes in cells and experimental animals therebyfacilitating functional analysis of the target or an appraisal of itsusefulness as a target for therapeutic intervention. In diagnostics theoligonucleotides may be used to detect and quantitate target expressionin cell and tissues by Northern blotting, in-situ hybridisation orsimilar techniques. For therapeutics, an animal or a human, suspected ofhaving a disease or disorder, which can be treated by modulating theexpression of target is treated by administering the oligonucleotidecompounds in accordance with this invention. Further provided aremethods of treating an animal particular mouse and rat and treating ahuman, suspected of having or being prone to a disease or condition,associated with expression of target by administering a therapeuticallyor prophylactically effective amount of one or more of theoligonucleotide compounds or compositions of the invention.

Therapeutic Use of Oligonucleotides Targeting miR-122a

We have demonstrated that a LNA-antimiR, targeting miR-122a reducesplasma cholesterol levels. Therefore, another aspect of the invention isuse of the above described oligonucleotides targeting miR-122a asmedicine.

Still another aspect of the invention is use of the above describedoligonucleotides targeting miR-122a for the preparation of a medicamentfor treatment of increased plasma cholesterol levels (orhypercholesterolemia and related disorders). The skilled man willappreciate that increased plasma cholesterol levels is undesirable as itincreases the risk of various conditions, e.g. atherosclerosis.

Still another aspect of the invention is use of the above describedoligonucleotides targeting miR-122a for upregulating the mRNA levels ofNrdg3, Aldo A, Bckdk or CD320.

Embodiments

The following embodiments of the present invention may be used incombination with the other embodiments described herein.

1. A pharmaceutical composition comprising an oligomer of between 6-12nucleotides in length, wherein said oligomer comprises a contiguousnucleotide sequence of a total of between 6-12 nucleotides, such as 6,7, 8, 9, 10, 11 or 12 nucleotide units, wherein at least 50% of thenucleobase units of the oligomer are high affinity nucleotide analogueunits, and a pharmaceutically acceptable diluent, carrier, salt oradjuvant.2. The pharmaceutical composition according to embodiment 1, wherein thecontiguous nucleotide sequence is complementary to a correspondingregion of a mammalian, human or viral microRNA (miRNA) sequence.3. The pharmaceutical composition according to embodiment 2, wherein thecontiguous nucleotide sequence is complementary to a correspondingregion of a miRNA sequence selected from the group of miRNAs listed inany one of tables 3, 4 or 5.4. The pharmaceutical composition according to embodiment 2 or 3,wherein the contiguous nucleotide sequence consists of or comprises asequence which is complementary to the seed sequence of said microRNA.5. The pharmaceutical composition according to any one of embodiments2-4, wherein the contiguous nucleotide sequence consists of or comprisesa sequence selected from any one of the sequences listed in table 3 or4.6. The pharmaceutical composition according to embodiment 4 or 5,wherein the 3′ nucleobase of the seedmer forms the 3′ most nucleobase ofthe contiguous nucleotide sequence, wherein the contiguous nucleotidesequence may, optionally, comprise one or two further 5′ nucleobases.7. The pharmaceutical composition according to any one of embodiments1-6, wherein said contiguous nucleotide sequence does not comprise anucleotide which corresponds to the first nucleotide present in themicro RNA sequence counted from the 5′ end.8. The pharmaceutical composition according to any one of embodiments1-7, wherein the contiguous nucleotide sequence is complementary to acorresponding nucleotide sequence present in a miRNA selected from thoseshown in table 3 or 4 or 5.9. The pharmaceutical composition according to embodiment 8, whereinsaid miRNA is selected from the group consisting of miR-1, miR-10b,miR-17-3p, miR-18, miR-19a, miR-19b, miR-20, miR-21, miR-34a, miR-93,miR-106a, miR-106b, miR-122, miR-133, miR-134, miR-138, miR-155,miR-192, miR-194, miR-221, miR-222, and miR-375.10. The pharmaceutical composition according to any one of embodiments1-9, wherein at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or all ofthe nucleobase units of the contiguous nucleotide sequence arenucleotide analogue units.11. The pharmaceutical composition according to embodiment 10, whereinthe nucleotide analogue units are selected from the group consisting of2′-O_alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNAunit, LNA unit, PNA unit, HNA unit, INA unit, and a 2′MOE RNA unit.12. The pharmaceutical composition according to embodiment 10 or 11,wherein at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or all of thenucleobase units of the contiguous nucleotide sequence are LockedNucleic Acid (LNA) nucleobase units.13. The pharmaceutical composition according to embodiment 12, whereinall of the nucleobase units of the contiguous nucleotide sequence areLNA nucleobase units.14. The pharmaceutical composition according to any one of embodiments1-13, wherein the contiguous nucleotide sequence comprises or consistsof 7, 8, 9 or 10, preferably contiguous, LNA nucleobase units.15. The pharmaceutical composition according to any one of embodiments1-14, wherein the oligomer consist of 7, 8, 9 or 10 contiguousnucleobase units and wherein at least 7 nucleobase units are nucleotideanalogue units.16. The pharmaceutical composition according to embodiment 15, whereinthe nucleotide analogue units are Locked Nucleic Acid (LNA) nucleobaseunits.17. The pharmaceutical composition according to embodiment 15, whereinthe nucleotide analogue units in the molecule consists of a mixture ofat least 50% LNA units and up to 50% other nucleotide analogue units.18. The pharmaceutical composition according to any one of embodiments1-17, wherein at least 75%, such as 80% or 85% or 90% or 95% or all ofthe internucleoside linkages present between the nucleobase units of thecontiguous nucleotide sequence are phosphorothioate internucleosidelinkages.19. The pharmaceutical composition according to any one of embodiments1-18, wherein said oligomer is conjugated with one or morenon-nucleobase compounds.20. The pharmaceutical composition according to any one of embodiments1-19, wherein the contiguous nucleotide sequence is complementary to thecorresponding sequence of at least two miRNA sequences such as 2, 3, 4,5, 6, 7, 8, 9, or 10 miRNA sequences.21. The pharmaceutical composition according to any one of embodiments1-20, wherein the contiguous nucleotide sequence consists or comprisesof a sequence which is complementary to the sequence of at least twomiRNA seed region sequences such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 miRNAseed region sequences.22. The pharmaceutical composition according to any one of embodiments20 or 21, wherein the contiguous nucleotide sequence is complementary tothe corresponding region of both miR-221 and miR-222.23. The pharmaceutical composition according to embodiment 22, whereinthe contiguous nucleotide sequence consists or comprises of a sequencethat is complementary to 5′GCUACAU3′.24. The pharmaceutical composition according to any one of embodiments1-23, wherein the oligomer is constituted as a prodrug.25. The pharmaceutical composition according to any one of embodiments1-24, wherein the contiguous nucleotide sequence is complementary to acorresponding region of has-miR-122.26. The pharmaceutical composition according to embodiment 25, for usein the treatment of a medical disorder or disease selected from thegroup consisting of: hepatitis C virus infection andhypercholesterolemia and related disorders.27. The pharmaceutical composition according to embodiment 25 or 26,wherein the composition further comprises a second independent activeingredient that is an inhibitor of the VLDL assembly pathway, such as anApoB inhibitor, or an MTP inhibitor.28. A kit comprising a pharmaceutical composition according toembodiment 25 or 26, and a second independent active ingredient that isan inhibitor of the VLDL assembly pathway, such as an ApoB inhibitor, oran MTP inhibitor.29. A method for the treatment of a disease or medical disorderassociated with the presence or overexpression of a microRNA, comprisingthe step of administering a the pharmaceutical composition) according toany one of embodiments 1-28 to a patient who is suffering from, or islikely to suffer from said disease or medical disorder.30. An oligomer, as defined according to anyone of embodiments 1-25.31. A conjugate comprising the oligomer according to embodiment 30, andat least one non-nucleobase compounds.32. The use of an oligomer or a conjugate as defined in any one ofembodiments 30-31, for the manufacture of a medicament for the treatmentof a disease or medical disorder associated with the presence orover-expression of the microRNA.33. A method for reducing the amount, or effective amount, of a miRNA ina cell, comprising administering an oligomer, a conjugate or apharmaceutical composition, according to any one of the preceedingembodiments to the cell which is expressing said miRNA so as to reducethe amount, or effective amount of the miRNA in the cell.34. A method for de-repression of a mRNA whose expression is repressedby a miRNA in a cell comprising administering an oligomer, a conjugateor a pharmaceutical composition, according to any one of the preceedingembodiments to the cell to the cell which expressed both said mRNA andsaid miRNA, in order to de-repress the expression of the mRNA.

References

Details of the reference are provided in the priority documents.

EXAMPLES

LNA Monomer and oligonucleotide synthesis were performed using themethodology referred to in Examples 1 and 2 of WO2007/112754. Thestability of LNA oligonucleotides in human or rat plasma is performedusing the methodology referred to in Example 4 of WO2007/112754. Thetreatment of in vitro cells with LNA anti-miR antisense oligonucleotide(targeting miR-122) is performed using the methodology referred to inExample 6 of WO2007/112754. The analysis of Oligonucleotide Inhibitionof miR expression by microRNA specific quantitative PCR in both an invitro and in vivo model is performed using the methodology referred toin Example 7 of WO2007/112754. The assessment of LNA antimir knock-downspecificity using miRNA microarray expression profiling is performedusing the methodology referred to in Example 8 of WO2007/112754. Thedetection of microRNAs by in situ hybridization is performed using themethodology referred to in Example 9 of WO2007/112754. The Isolation andanalysis of mRNA expression (total RNA isolation and cDNA synthesis formRNA analysis) in both an in vitro and in vivo model is performed usingthe methodology referred to in Example 10 of WO2007/112754. In vivoExperiments using Oligomers of the invention targeting microRNA-122, andsubsequent analysis are performed using the methods disclosed inExamples 11-27 of WO2007/112754. The above mentioned examples ofWO2007/112754 are hereby specifically incorporated by reference.

Example 1 Design of the LNA antimiR Oligonucleotides and MeltingTemperatures

TABLE 2 Oligomers used in the examples and figures. The Compound numberis an identifier used throughout the examples and figures - theSEQ ID NO which is used in the sequence listing is also provided. SEQ IDCompound NO Compound Sequence Comment 3204 1 TcAGtCTGaTaAgCT 3205 2GATAAGCT 3206 3 TcAcAATtaGCAtTA 3207 4 TAGCATTA 4 5 CcAttGTcaCaCtCC 32086 CACACTCC 3209 7 TAAGCT 3210 8 ATAAGCT 3211 9 TGATAAGCT 3212 10CTGATAAGCT 3213 11 GTCTGATAAGCT 3214 12 CAGTCTGATAAGCT 3215 13 TCTGATAA3216 14 ATCAGTCT 3217 15 TCAACATC 3218 or 3230 16 G G TAA A CTUnderline = mismatch 3219 17 CG TAA TGA Underline = mismatch 3220 18TCAgtctgataaGCTa 5′ fluorescent label (FAM) 3221 19 AGCACTTT 3222 20ATTTGCAC 3223 21 AgCagACaaTgTaGC 5′ fluorescent label (FAM) 3224 22GtAgcCAgaTgTaGC 5′ fluorescent label (FAM) 3225 23 ATGTAGC 3226 24ACaAcCTacTaCcTC 3227 25 ACTACCTC 3228 26 CaCtgTCagCaCtTT 3229 27TgCatAGatTtGcAC 3231 28 GTAGACT 3232 29 TACCTC 3233 30 CTACCTC 3234 31TNCTACCTC N = universal base. 3235 32 TNCTACCTC N = universal base. 323633 GCaAcCTacTaCcTC 3237 34 ACaAcCTccTaCcTC 3238 35 ACaAaCTacTaCcTC 323936 CTACCTC 3240 37 CTAACTC 3241 38 TTAGCATTA 3242 39 CGATTAGCATTA 3243977 CACGATTAGCATTA 3244 978 GCATTA 3245 979 AGCATTA 3246 980 ATTAGCATTACapital and lower case letters denote LNA and DNA, respectively. LNAcytosines are preferably methyl cytosine/5′methyl-cytosine* Allinternucleoside linkages are preferably phosphorothioate* All LNA may,for example, be beta-D-oxy LNA* *Used in the specific examples,

Example 2 In Vitro Model: Cell Culture

The effect of LNA oligonucleotides on target nucleic acid expression(amount) can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. Target can beexpressed endogenously or by transient or stable transfection of anucleic acid encoding said nucleic acid.

The expression level of target nucleic acid can be routinely determinedusing, for example, Northern blot analysis (including microRNAnorthern), Quantitative PCR (including microRNA qPCR), Ribonucleaseprotection assays. The following cell types are provided forillustrative purposes, but other cell types can be routinely used,provided that the target is expressed in the cell type chosen.

Cells were cultured in the appropriate medium as described below andmaintained at 37° C. at 95-98% humidity and 5% CO₂. Cells were routinelypassaged 2-3 times weekly.

15PC3: The human prostate cancer cell line 15PC3 was kindly donated byDr. F. Baas, Neurozintuigen Laboratory, AMC, The Netherlands and wascultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+GlutamaxI+gentamicin.PC3: The human prostate cancer cell line PC3 was purchased from ATCC andwas cultured in F12 Coon's with glutamine (Gibco)+10% FBS+gentamicin.518A2: The human melanoma cancer cell line 518A2 was kindly donated byDr. B. Jansen, Section of experimental Oncology, Molecular Pharmacology,Department of Clinical Pharmacology, University of Vienna and wascultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+GlutamaxI+gentamicin.HeLa: The cervical carcinoma cell line HeLa was cultured in MEM (Sigma)containing 10% fetal bovine serum gentamicin at 37° C., 95% humidity and5% CO₂.MP-11: The murine multiple myeloma cell line MPC-11 was purchased fromATCC and maintained in DMEM with 4 mM Glutamax+10% Horse Serum.DU-145: The human prostate cancer cell line DU-145 was purchased fromATCC and maintained in RPMI with Glutamax+10% FBS.RCC-4+/−VHL: The human renal cancer cell line RCC4 stably transfectedwith plasmid expressing VHL or empty plasmid was purchased from ECACCand maintained according to manufacturers instructions.786-0: The human renal cell carcinoma cell line 786-0 was purchased fromATCC and maintained according to manufacturers instructionsHUVEC: The human umbilical vein endothelial cell line HUVEC waspurchased from Camcrex and maintained in EGM-2 medium.K562: The human chronic myelogenous leukaemia cell line K562 waspurchased from ECACC and maintained in RPMI with Glutamax+10% FBS.U87MG: The human glioblastoma cell line U87MG was purchased from ATCCand maintained according to the manufacturers instructions.B16: The murine melanoma cell line B16 was purchased from ATCC andmaintained according to the manufacturers instructions.LNCap: The human prostate cancer cell line LNCap was purchased from ATCCand maintained in RPMI with Glutamax+10% FBSHuh-7: Human liver, epithelial like cultivated in Eagles MEM with 10%FBS, 2 mM Glutamax I, 1× non-essential amino acids, Gentamicin 25 μg/mlL428: (Deutsche Sammlung für Mikroorganismen (DSM, Braunschwieg,Germany)): Human B cell lymphoma maintained in RPMI 1640 supplementedwith 10% FCS, L-glutamine and antibiotics.L1236: (Deutsche Sammlung für Mikroorganismen (DSM, Braunschwieg,Germany)): Human B cell lymphoma maintained in RPMI 1640 supplementedwith 10% FCS, L-glutamine and antibiotics.

Example 3 Design of a LNA antimiR Library for all Human microRNASequences in miRBase microRNA Database

The miRBase version used was version 12, as reported in Griffiths-Jones,S., Grocock, R. J., van Dongen, S., Bateman, A., Enright, A. J. 2006.miRBase: microRNA sequences, targets and gene nomenclature. NucleicAcids Res. 34: 0140-4, and available viahttp://microrna.sanger.ac.uk/sequences/index.shtml.

Table 1 shows 7-, 8- and 9-mer nucleotide sequences comprising theseedmer sequence of micro RNA's according to the miRBase micro RNAdatabase. The seedmer sequence comprises the reverse complement of themicroRNA seed region. In some embodiments the oligomer of the inventionhas a contiguous nucleotide sequence selected from the 7-mer, 8-mer or9-mer sequences. With respect to the 7-mer, 8-mer and 9-mer sequences,in some embodiments, all the internucleoside linkages arephosphorothioate. The 7-mer, 8-mer and 9-mer nucleotide sequences mayconsist of sequence of nucleotide analogues as described herein, such asLNA nucleotide analogues. LNA cytosines may be methyl-cytosine(5′methyl-cytosine). In some embodiments, the LNA is beta-D-oxy-LNA.

Table 3 provides a list of microRNAs grouped into those which can betargeted by the same seedmer oligomers, such as the 7-, 8- or 9-mersprovided herein (see table 1).

TABLE 3 hsa-let-7a* (SEQ ID NO: 97), hsa-let-7f-1* (SEQ ID NO: 107)hsa-let-7a (SEQ ID NO: 96), hsa-let-7b (SEQ ID NO: 98), hsa-let-7c (SEQID NO: 100), hsa-let-7d (SEQ ID NO: 102), hsa-let-7f (SEQ ID NO: 106),hsa-miR-98 (SEQ ID NO: 947), hsa-let-7g (SEQ ID NO: 109), hsa-let-7i(SEQ ID NO: 111) hsa-miR-1 (SEQ ID NO: 113), hsa-miR-206 (SEQ ID NO:403) hsa-miR-103 (SEQ ID NO: 118), hsa-miR-107 (SEQ ID NO: 126)hsa-miR-10a (SEQ ID NO: 127), hsa-miR-10b (SEQ ID NO: 129) hsa-miR-125b(SEQ ID NO: 190), hsa-miR-125a-5p (SEQ ID NO: 189) hsa-miR-129* (SEQ IDNO: 229), hsa-miR-129-3p (SEQ ID NO: 230) hsa-miR-130a (SEQ ID NO: 251),hsa-miR-301a (SEQ ID NO: 473), hsa-miR-130b (SEQ ID NO: 253),hsa-miR-454 (SEQ ID NO: 608), hsa-miR-301b (SEQ ID NO: 474) hsa-miR-133a(SEQ ID NO: 261), hsa-miR-133b (SEQ ID NO: 262) hsa-miR-135a (SEQ ID NO:264), hsa-miR-135b (SEQ ID NO: 266) hsa-miR-141 (SEQ ID NO: 278),hsa-miR-200a (SEQ ID NO: 392) hsa-miR-146a (SEQ ID NO: 290),hsa-miR-146b-5p (SEQ ID NO: 293) hsa-miR-152 (SEQ ID NO: 308),hsa-miR-148b (SEQ ID NO: 300) hsa-miR-154* (SEQ ID NO: 314),hsa-miR-487a (SEQ ID NO: 619) hsa-miR-15a (SEQ ID NO: 317), hsa-miR-16(SEQ ID NO: 321), hsa-miR-15b (SEQ ID NO: 319), hsa-miR-195 (SEQ ID NO:377), hsa-miR-497 (SEQ ID NO: 634) hsa-miR-17 (SEQ ID NO: 324),hsa-miR-20a (SEQ ID NO: 406), hsa-miR-93 (SEQ ID NO: 930), hsa-miR-106a(SEQ ID NO: 122), hsa-miR-106b (SEQ ID NO: 124), hsa-miR-20b (SEQ ID NO:408), hsa-miR-526b* (SEQ ID NO: 708) hsa-miR-181a (SEQ ID NO: 326),hsa-miR-181c (SEQ ID NO: 330) hsa-miR-181b (SEQ ID NO: 329),hsa-miR-181d (SEQ ID NO: 332) hsa-miR-18a (SEQ ID NO: 349), hsa-miR-18b(SEQ ID NO: 351) hsa-miR-190 (SEQ ID NO: 353), hsa-miR-190b (SEQ ID NO:357) hsa-miR-192 (SEQ ID NO: 369), hsa-miR-215 (SEQ ID NO: 417)hsa-miR-196a (SEQ ID NO: 379), has-miR-196b (SEQ ID NO: 381)hsa-miR-199a-3p, hsa-miR-199b-3p (SEQ ID NO: 385) hsa-miR-199a-5p (SEQID NO: 384), hsa-miR-199b-5p (SEQ ID NO: 386) hsa-miR-19a* (SEQ ID NO:388), hsa-miR-19b-1* (SEQ ID NO: 390), hsa-miR-19b-2* (SEQ ID NO: 391)hsa-miR-19a (SEQ ID NO: 387), hsa-miR-19b (SEQ ID NO: 389) hsa-miR-200b(SEQ ID NO: 394), has-miR-200c (SEQ ID NO: 396) hsamiR-204 (SEQ ID NO:401), hsa-miR-211 (SEQ ID NO: 413) hsa-miR-208a (SEQ ID NO: 404),hsa-miR-208b (SEQ ID NO: 405) hsa-miR-212 (SEQ ID NO: 414), hsa-miR-132(SEQ ID NO: 255) hsa-miR-23a* (SEQ ID NO: 440), hsa-miR-23b*(SEQ ID NO:442) hsa-miR-23a (SEQ ID NO: 439), hsa-miR-23b (SEQ ID NO: 441),hsa-miR-130a* (SEQ ID NO: 252) hsa-miR-24-1* (SEQ ID NO: 444),hsa-miR-24-2* (SEQ ID NO: 445) hsa-miR-25 (SEQ ID NO: 446), hsa-miR-92a(SEQ ID NO: 925), hsa-miR-367 (SEQ ID NO: 547), hsa-miR-92b (SEQ ID NO:928) hsa-miR-26a (SEQ ID NO: 448), hsa-miR-26b (SEQ ID NO: 451)hsa-miR-26a-1* (SEQ ID NO: 449), hsa-miR-26a-2* (SEQ ID NO: 450)hsa-miR-27a (SEQ ID NO: 453), hsa-miR-27b (SEQ ID NO: 455) hsa-miR-29a(SEQ ID NO: 465), hsa-miR-29b (SEQ ID NO: 467), hsa-miR-29c (SEQ ID NO:470) hsa-miR-302a (SEQ ID NO: 475), hsa-miR-302b (SEQ ID NO: 477),hsa-miR-302c (SEQ ID NO: 479), hsa-miR-302d (SEQ ID NO: 481),hsa-miR-373 (SEQ ID NO: 555), hsa-miR-520e (SEQ ID NO: 696),hsa-miR-520a-3p (SEQ ID NO: 690), hsa-miR-520b (SEQ ID NO: 692),hsa-miR-520c-3p (SEQ ID NO: 693), hsa-miR-520d-3p (SEQ ID NO: 694)hsa-miR-302b* (SEQ ID NO: 478), hsa-miR-302d* (SEQ ID NO: 482)hsa-miR-30a* (SEQ ID NO: 486), hsa-miR-30d* (SEQ ID NO: 493),hsa-miR-30e* (SEQ ID NO: 495) hsa-miR-30a (SEQ ID NO: 485), hsa-miR-30c(SEQ ID NO: 489), hsa-miR-30d (SEQ ID NO: 492), hsa-miR-30b (SEQ ID NO:487), hsa-miR-30e (SEQ ID NO: 494) hsa-miR-330-5p (SEQ ID NO: 513),hsa-miR-326 (SEQ ID NO: 509) hsa-miR-34a (SEQ ID NO: 534),hsa-miR-34c-5p (SEQ ID NO: 539), hsa-miR-449a (SEQ ID NO: 599),hsa-miR-449b (SEQ ID NO: 600) hsa-miR-362-3p (SEQ ID NO: 542),hsa-miR-329 (SEQ ID NO: 511) hsa-miR-374a (SEQ ID NO: 557), hsa-miR-374b(SEQ ID NO: 559) hsa-miR-376a (SEQ ID NO: 562), hsa-miR-376b (SEQ ID NO:564) hsa-miR-378 (SEQ ID NO: 568), hsa-miR-422a (SEQ ID NO: 585)hsa-miR-379* (SEQ ID NO: 571), hsa-miR-411* (SEQ ID NO: 582) hsa-miR-381(SEQ ID NO: 574), hsa-miR-300 (SEQ ID NO: 472) hsa-miR-509-5p (SEQ IDNO: 655), hsa-miR-509-3-5p (SEQ ID NO: 653) hsa-miR-515-5p (SEQ ID NO:666), hsa-miR-519e* (SEQ ID NO: 689) hsa-miR-516b*, hsa-miR-516a-3p (SEQID NO: 667) hsa-miR-517a (SEQ ID NO: 671), hsa-miR-517c (SEQ ID NO: 673)hsa-miR-518a-5p (SEQ ID NO: 674), hsa-miR-527 (SEQ ID NO: 709)hsa-miR-518f (SEQ ID NO: 681), hsa-miR-518b (SEQ ID NO: 675),hsa-miR-518c (SEQ ID NO: 676), hsa-miR-518a-3p (SEQ ID NO: 674),hsa-miR-518d-3p (SEQ ID NO: 678) hsa-miR-519c-3p (SEQ ID NO: 686),hsa-miR-519b-3p (SEQ ID NO: 685), hsa-miR-519a (SEQ ID NO: 683)hsa-miR-519c-5p, hsa-miR-519b-5p, hsa-miR-523*, hsa-miR-518f* (SEQ IDNO: 682), hsa-miR-526a, hsa-miR-520c-5p, hsa-miR-518e*, hsa-miR-518d-5p(SEQ ID NO: 679), hsa-miR-522*, hsa-miR-519a* (SEQ ID NO: 684)hsa-miR-519e (SEQ ID NO: 688), hsa-miR-33b* (SEQ ID NO: 527)hsa-miR-520a-5p (SEQ ID NO: 691), hsa-miR-525-5p (SEQ ID NO: 706)hsa-miR-520g (SEQ ID NO: 698), hsa-miR-520h (SEQ ID NO: 699)hsa-miR-524-5p (SEQ ID NO: 704), hsa-miR-520d-5p (SEQ ID NO: 695)hsa-miR-525-3p (SEQ ID NO: 705), hsa-miR-524-3p (SEQ ID NO: 703)hsa-miR-548b-5p (SEQ ID NO: 724), hsa-miR-548a-5p (SEQ ID NO: 722),hsa-miR-548c-5p (SEQ ID NO: 726), hsa-miR-548d-5p (SEQ ID NO: 728)hsa-miR-7-1* (SEQ ID NO: 880), hsa-miR-7-2* (SEQ ID NO: 881) hsa-miR-99a(SEQ ID NO: 948), hsa-miR-100 (SEQ ID NO: 114), hsa-miR-99b (SEQ ID NO:950)

We have constructed an 8-mer LNA-antimiR against miR-21, miR-155 andmiR-122 (designated here as micromiR) that is fully LNA modified andphosphorothiolated (see FIG. 1 and Table 6). Our results from repeatedexperiments in MCF-7, HeLa, Raw and Huh-7 cells using a luciferasesensor plasmid for miR-21, miR-155 and miR-122 demonstrate that thefully LNA-modified short LNA-antimiRs are highly potent in antagonizingmicroRNAs.

TABLE 4 LNA_antimiR & MicromiR sequences and predicted T_(m)s SEQ T_(m)Compound ID NO microRNA Sequence (° C.) 3204 1 miR-21 T c A G t C T G aT a 73 A g C T 3205 2 G A T A A G C T 33 3206 3 miR-155 T c A c A A T ta 63 G C A t T A 3207 4 T A G C A T T A 45 4 5 mRi22 C c A t t G T c a C73 a C t C C 3208 6 C A C A C T C C 62 Capital letters are LNA units,such as beta-D-oxy LNA. Lower case letters are DNA units.Internucleoside linkages are preferably phosphorothioate. LNA cytosinesare all preferably methylated/5-methyl cytosine.

The melting temperatures can be assessed towards the mature microRNAsequence, using a synthetic microRNA oligonucleotide (typicallyconsisting of RNA nucleotides with a phosphodiester backbone). Typicallymeasured T_(m)s are higher than predicted Tins when using LNA oligomersagainst the RNA target.

Example 4 Assessment of miR-21 Antagonism by Compound 3205 (SEQ ID NO:2) LNA-antimiR in MCF-7 Cells Using a Luciferase Sensor Assay

In order to assess the efficiency of a fully LNA-modified 8-merLNA-antimiR (Compound 3205, SEQ ID NO: 2) oligonucleotide in targetingand antagonizing miR-21, luciferase sensor constructs were madecontaining a perfect match target site for the mature miR-21 and ascontrol, a target site with two mutations in the seed (FIG. 6). In orderto monitor microRNA-21 inhibition, the breast carcinoma cell line MCF-7was transfected with the different luciferase constructs together withthe miR-21 antagonist Compound 3205 (SEQ ID NO: 2) at varyingconcentrations in comparison with a 15-mer LNA-antimiR Compound 3204(SEQ ID NO: 1) against miR-21. After 24 hours, luciferase activity wasmeasured.

Results:

As seen in FIG. 2, the new fully LNA-modified 8-mer LNA-antimiR(Compound 3205 (SEQ ID NO: 2) shows two-fold higher potency compared toCompound 3204 (SEQ ID NO: 1), as shown by de-repression of theluciferase activity. By contrast, the control miR-21 sensor constructwith two mismatches in the miR-21 seed did not show any de-repression ofthe firefly luciferase activity, thereby demonstrating the specificityof the perfect match miR-21 sensor in monitoring miR-21 activity incells. The de-repression of luciferase activity by the 8-mer LNA-antimiRis dearly dose-dependent, which is not seen with Compound 3204 (SEQ IDNO: 1). Moreover, the new 8-mer is also much more potent at lower dosesthan Compound 3204 (SEQ ID NO: 1).

To conclude, the 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2) showssignificantly improved potency in inhibition of miR-21 in vitro comparedto the 15-mer LNA-antimiR Compound 3204 (SEQ ID NO: 1) targeting miR-21.

Materials and Methods:

Cell Line:

The breast carcinoma cell line MCF-7 was purchased from ATCC (#HTB-22™).MCF-7 cells were cultured in EMEM medium, supplemented with 10% fetalbovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

400.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, MCF-7 cells were transfected with 0.8 ug miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vector(SDS Promega) together with 1 μl Lipofectamine2000 (Invitrogen)according to manufacturer's instructions. After 24 hours, cells wereharvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and harvested with cell scraper, afterwhich cells were centrifugated for 5 min at 10.000 rpm. The supernatantwas discarded and 50 μl 1× Passive Lysis Buffer (Promega) was added tothe cell pellet, after which cells were put on ice for 30 min. The lysedcells were spinned at 10.000 rpm for 30 min after which 20 μl weretransferred to a 96 well plate and luciferase measurements wereperformed according to manufacturer's instructions (Promega).

Example 5 Assessment of miR-21 Antagonism by Compound 3205 (SEQ ID NO:2) LNA-antimiR in HeLa Cells Using a Luciferase Sensor Assay

To further assess the efficiency of the fully LNA-modified 8-merLNA-antimiR Compound 3205 (SEQ ID NO: 2) in targeting miR-21, the cervixcarcinoma cell line HeLa was also transfected with the previouslydescribed miR-21 luciferase sensor constructs alongside Compound 3205(SEQ ID NO: 2) at varying concentrations as described in the abovesection (FIG. 3).

Results:

Compound 3205 (SEQ ID NO: 2) shows complete de-repression of the miR-21luciferase sensor construct in HeLa cells already at 5 nM compared toCompound 3204 (SEQ ID NO: 1), which did not show complete de-repressionuntil the highest dose (50 nM). In addition, antagonism of miR-21 by the8-mer Compound 3205 (SEQ ID NO: 2) LNA-antimiR is dose-dependent. Todemonstrate the specificity of the miR-21 luciferase sensor assay, amismatched miR-21 target site (2 mismatches in seed) was alsotransfected into HeLa cells, but did not show any de-repression of thefirefly luciferase activity.

To conclude, the fully LNA-modified Compound 3205 (SEQ ID NO: 2) showssignificantly improved potency in inhibition of miR-21 in vitro, in bothMCF-7 and HeLa cells compared to the 15-mer LNA-antimiR Compound 3204(SEQ ID NO: 1).

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24 well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 0.2 ug miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vectortogether with 0.7 μl Lipofectamine2000 (Invitrogen) according tomanufacturer's instructions. After 24 hours, cells were harvested forluciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24 well plates was puton an orbital shaker for 30 min. The cells were collected andtransferred to an eppendorf tube and spinned at 10.000 rpm for 30 minafter which 10 μl were transferred to a 96 well plate and luciferasemeasurements were performed according to manufacturer's instructions(Promega).

Example 6 Assessment of miR-155 Antagonism by Compound 3207 (SEQ ID NO:4) LNA-antimiR in Mouse RAW Cells Using a Luciferase Sensor Assay

To ask whether a fully LNA-modified 8-mer LNA-antimiR can effectivelyantagonize miR-155, a perfect match target site for miR-155 was clonedinto the same luciferase vector (psiCHECK2) and transfected into themouse leukaemic monocyte macrophage RAW cell line. Because theendogenous levels of miR-155 are low in the RAW cell line, the cellswere treated with 100 ng/ml LPS for 24 hours in order to induce miR-155accumulation.

Results:

Luciferase measurements showed that the fully LNA-modified 8-merLNA-antimiR Compound 3207 (SEQ ID NO: 4) targeting miR-155 was similarlyeffective in antagonizing miR-155 compared to the 15-mer LNA-antimiRCompound 3206 (SEQ ID NO: 3) (FIG. 4). Both LNA-antimirs showed a >50%de-repression of the miR-155 luciferase sensor at 0.25 nM concentrationand inhibited miR-155 in a dose-dependent manner.

Conclusion:

These data further support the results from antagonizing miR-21, asshown in examples 1 and 2, demonstrating that a fully thiolated 8-merLNA-antimiR is highly potent in microRNA targeting.

Materials and Methods:

Cell Line:

The mouse leukaemic monocyte macrophage RAW 264.7 was purchased fromATCC (TIB-71). RAW cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

500.000 cells were seeded per well in a 6 well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection. MCF-7 cells were transfected with 0.3 ug miR-155 orempty psiCHECK2 vector together with 10 μl Lipofectamine2000(Invitrogen) according to manufacturer's instructions. In order toinduce miR-155 accumulation, LPS (100 ng/ml) was added to the RAW cellsafter the 4 hour incubation with the transfection complexes. Afteranother 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and harvested with cell scraper, afterwhich cells were centrifugated for 5 min at 2.500 rpm. The supernatantwere discarded and 50 μl 1× Passive Lysis Buffer (Promega) was added tothe cell pellet, after which cells were put on ice for 30 min. The lysedcells were spinned at 10.000 rpm for 30 min after which 20 μl weretransferred to a 96 well plate and luciferase measurements wereperformed according to manufacturer's instructions (Promega).

Example 7 Assessment of miR-122 Antagonism by Compound 3208 (SEQ ID NO:6) LNA-antimiR in HuH-7 Cells Using a Luciferase Sensor Assay

The potency of the fully modified 8-mer LNA-antimiR Compound 3208 (SEQID NO: 6) against miR-122 was assessed in the human hepatoma cell lineHuH-7. The HuH-7 cells were transfected with luciferase sensor constructcontaining a perfect match miR-122 target site. After 24 hoursluciferase measurements were performed (FIG. 5).

Results:

The fully LNA-modified 8-mer LNA-antimiR Compound 3208 (SEQ ID NO: 6) ismore potent than the 15-mer LNA-antimiR Compound 4 (SEQ ID NO:5) at lowconcentration, as shown by de-repression of the miR-122 luciferasesensor. Both LNA-antimiRs inhibit miR-122 in a dose-dependent manner(FIG. 5).

Conclusion:

The fully LNA-modified 8-mer LNA-antimiR Compound 3208 (SEQ ID NO: 6)targeting miR-122 shows improved potency in inhibition of miR-122 invitro.

Materials and Methods:

Cell Line:

The human hepatoma cell line HuH-7 was a kind gift from R.Bartenschlager, Heidelberg. Huh-7 cells were cultured in EMEM medium,supplemented with 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25ug/ml Gentamicin.

Transfection:

8.000 cells were seeded per well in a 96 well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, HuH-7 cells were transfected with 57 ng miR-122 orempty psiCHECK2 vector together with 1 I Lipofectamine2000 (Invitrogen).After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

50 μl 1× Passive Lysis Buffer (Promega) was added to each well, afterwhich the 96 well plate was put on an orbital shaker for 30 min. To eachwell the Dual-luciferase Reporter assay system (Promega) was added andluciferase measurements were performed according to manufacturer'sinstructions (Promega).

Example 8 Assessment of miR-21 Antagonism by Comparing an 8-Mer(Compound 3205, SEQ ID NO: 2) Versus a 15-Mer (Compound 3204,SEQ IDNO: 1) LNA-antimiR in Human Prostate Carcinoma Cells (PC3)

We have previously shown (patent application 1051), that an 8-merLNA-antimiR that is fully LNA-modified and phosphorothiolated is able tocompletely de-repress the miR-21 luciferase reporter levels in the humancervix carcinoma cell line HeLa and partly de-repress the miR-21luciferase reporter levels in the human breast carcinoma cell lineMCF-7. We next extended this screening approach to the human prostatecancer cell line PC3. To assess the efficiency of the differentLNA-antimiR oligonucleotides against miR-21, luciferase reporterconstructs were generated in which a perfect match target site for themature miR-21 and a target site with two mismatches in the seed werecloned in the 3′UTR of Renilla luciferase gene (FIG. 7). In order tomonitor miR-21 inhibition, PC3 cells were transfected with the differentluciferase constructs together with the miR-21 antagonist Compound 3205(SEQ ID NO: 2) (8-mer) and for comparison with the 15-mer LNA-antimiRperfect match Compound 3204 (SEQ ID NO: 1) at varying concentrations.After 24 hours, luciferase activity was measured.

Results:

The luciferase reporter experiments showed a dose-dependentde-repression of the luciferase miR-21 reporter activity with the 15-merLNA-antimiR against miR-21 (Compound 3204, SEQ ID NO: 1). However,complete de-repression of the luciferase reporter was not obtained evenat the highest concentrations (FIG. 7). In contrast, the cells that weretransfected with the 8-mer fully LNA substituted LNA-antimiR showedcomplete de-repression already at 1 nM, indicating significantlyimproved potency compared to the 15-mer LNA-antimiR. The luciferasecontrol reporter harboring a mismatch target site for miR-21 was notaffected by either LNA-antimiR, demonstrating high specificity of bothLNA-antimiRs.

Conclusion:

The micromer is far more potent than the 15-mer LNA-antimiR in targetingmiR-21 and has so far shown to be most potent in prostate carcinomacells.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714). PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

100.000 cells were seeded per well in a 12-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, PC3 cells were transfected with 0.3 μg miR-21 or emptypsiCHECK2 vector together with 1.2 μl Lipofectamine2000 (Invitrogen)according to manufacturers instructions. Transfected was also varyingconcentrations of LNA-antimiRs. After 24 hours, cells were harvested forluciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 250 μl 1× Passive Lysis Buffer(Promega) was added to the wells. The plates were placed on a shaker for30 min., after which the cell lysates were transferred to eppendorftubes. The cell lysate was centrifugated for 10 min at 2.500 rpm afterwhich 20 μl were transferred to a 96 well plate and luciferasemeasurements were performed according to manufacturer's instructions(Promega).

Example 9 Specificity Assessment of miR-21 Antagonism by an 8-MerLNA-antimiR

To investigate the specificity of our short LNA-antimiR targetingmiR-21, we designed an 8-mer mismatch control LNA-antimiR (Compound3218, SEQ ID NO: 16) containing 2 mismatches in the seed recognitionsequence (see FIG. 8). The luciferase reporter constructs described inexample 1 were transfected into the human cervix carcinoma cell lineHeLa together with the LNA mismatch control oligo Compound 3218 (SEQ IDNO: 16) and its efficacy was compared with the 8-mer LNA-antimiR(Compound 3205,SEQ ID NO: 2) targeting miR-21. After 24 hours,luciferase activity was measured.

Results:

As shown in FIG. 8, transfection of the fully LNA-modified 8-merLNA-antimiR in HeLa cells resulted in complete de-repression of theluciferase miR-21 reporter already at 5 nM. In contrast, when the cellswere transfected with the 8-mer LNA mismatch control oligo, combinedwith the results obtained with the control miR-21 luciferase reporterhaving two mismatches in the miR-21 seed, these data demonstrate highspecificity of the fully LNA-substituted 8-mer LNA-antimiR in targetingmiR-21 in Hela cells.

Analysis of the miRBase microRNA sequence database showed that themiR-21 recognition sequence, of the LNA-antimiR Compound 3205 (SEQ IDNO: 2) is unique for microRNA-21. However, when decreasing the micromerlength to 7 nt, it is not specific for only miR-21, since ath-miR-844,mmu-miR-590-3p and has-miR-590-3p are also targeted.

Conclusion:

Exchanging two nucleotide positions within the 8-mer LNA-antimiR withtwo mismatching nucleotides completely abolished the antagonizingactivity of the LNA-antimiR for miR-21.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24-well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 0.2 ug miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vectortogether with 0.7 μl Lipofectamine2000 (Invitrogen) according tomanufacturer's instructions. Transfected was also varying concentrationsof LNA-antimiRs. After 24 hours, cells were harvested for luciferasemeasurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates wereput on an orbital shaker for 30 min. The cells were collected andtransferred to an eppendorf tube and spinned at 10.000 rpm for 30 minafter which 10 μl were transferred to a 96-well plate and luciferasemeasurements were performed according to manufacturer's instructions(Promega).

Example 10 Assessment of the Shortest Possible Length of a FullyLNA-Modified LNA-antimiR that Mediates Effective Antagonism of miR-21

To further investigate the LNA-antimiR length requirements, we designeda 7-mer and a 6-mer LNA-antimiR targeting miR-21, both fullyLNA-modified and phosphorothiolated oligonucleotides. The miR-21luciferase reporter constructs were transfected into HeLa cells alongwith the LNA-antimiRs at varying concentrations. Luciferase measurementswere performed after 24 hours.

Results:

As seen in FIG. 9, the 7-mer LNA-antimiR mediates de-repression of themiR-21 luciferase reporter plasmid, but at lower potency compared to the8-mer LNA-antimiR (Compound 3205, SEQ ID NO: 2). Nevertheless, adose-dependent trend can still be observed. By contrast, the 6-merLNA-antimiR did not show any inhibitory activity.

Conclusion:

To conclude, the shortest possible length of an LNA-antimiR which isable to mediate miR-21 inhibition is 7 nucleotides. However, the 7-merLNA-antimiR is less potent compared to the 8-mer LNA-antimiR for miR-21.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24 well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 0.2 ug miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vectortogether with 0.7 μl Lipofectamine2000 (Invitrogen) according tomanufacturer's instructions. Transfected was also varying concentrationsof LNA-antimiRs. After 24 hours, cells were harvested for luciferasemeasurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates was puton an orbital shaker for 30 min. The cells were collected andtransferred to an eppendorf tube and spinned at 10.000 rpm for 30 minafter which 10 μl were transferred to a 96-well plate and luciferasemeasurements were performed according to manufacturers instructions(Promega).

Example 11 Length Assessment of Fully LNA-Substituted LNA-antimiRsAntagonizing miR-21

Next, we investigated the effect of increasing the length from a 9-merto a 14-mer fully LNA substituted LNA-antimiRs on antagonizing miR-21 inHeLa cells. The resulting LNA-antimiRs were transfected into HeLa cellstogether with the miR-21 luciferase reporter constructs (FIG. 10).Luciferase measurements were performed after 24 hours.

Results:

The 9-mer LNA-antimiR Compound 3211 (SEQ ID NO: 9) (9-mer) showeddose-dependent de-repression of the miR-21 luciferase reporter which didnot reach complete de-repression, as demonstrated for the 7-merLNA-antimiR (Compound 3210, SEQ ID NO: 8). Increasing the length to10-mer to 14-mer (Compound 3212, (SEQ ID NO: 10); Compound 3213 (SEQ IDNO: 11); and Compound 3214 (SEQ ID NO: 12)) decreased the potency asshown by less efficient de-repression of the miR-21 reporter.

Conclusion:

As shown in FIG. 10, the longest fully LNA-modified andphosphorothiolated LNA-antimiR which is still able to mediate miR-21inhibition is a 9-mer LNA-antimiR Compound 3211 (SEQ ID NO: 9). However,it is clearly less efficient than the 7-mer and 8-mer LNA-antimiRs.

Materials and Methods:

Cell Line: The human cervix carcinoma cell line HeLa was purchased fromECACC (#93021013). HeLa cells were cultured in EMEM medium, supplementedwith 10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/mlGentamicin.

Transfection:

60.000 cells were seeded per well in a 24-well plate the day beforetransfection in order to achieve 50-70% confluency the next day. On theday of transfection. HeLa cells were transfected with 0.2 ug miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 controlvector without target site together with 0.7 μl Lipofectamine2000(Invitrogen) according to manufacturer's instructions. Transfected wasalso varying concentrations of LNA-antimiRs. After 24 hours, cells wereharvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates wereput on an orbital shaker for 30 min. The cells were collected andtransferred to an eppendorf tube and spinned at 10.000 rpm for 30 minafter which 10 μl were transferred to a 96-well plate and luciferasemeasurements were performed according to manufacturer's instructions(Promega).

Example 12 Determination of the Most Optimal Position for an 8-MerLNA-antimiR within the miR Target Recognition Sequence

Our experiments have shown that the most potent fully LNA-modifiedphosphorothiolated LNA-antimiR is 8 nucleotides in length. To assess themost optimal position for an 8-mer LNA-antimiR within the miR targetrecognition sequence, we designed four different fully LNA-modified8-mer LNA-antimiRs tiled across the mature miR-21 sequence as shown inFIG. 11. The different LNA-antimiRs were co-transfected together withthe miR-21 luciferase reporter constructs into HeLa cells. Luciferasemeasurements were performed after 24 hours.

Results:

The only LNA-antimiR that mediated efficient silencing of miR-21 asmeasured by the luciferase reporter was Compound 3205 (SEQ ID NO: 2),which targets the seed region of miR-21. Neither Compound 3215 (SEQ IDNO: 13) which was designed to cover the 3′end of the seed (50% seedtargeting) did not show any effect, nor did the other two LNA-antimiRsCompound 3216 (SEQ ID NO: 14) or Compound 3217 (SEQ ID NO: 15), whichwere positioned to target the central region and the 3′end of the maturemiR-21, respectively.

Conclusion:

The only 8-mer LNA-antimiR mediating potent silencing of miR-21 is theone targeting the seed of the miR-21.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24-well plate the day beforetransfection in order to achieve 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 0.2 ug miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vectortogether with 0.7 d Lipofectamine2000 (Invitrogen) according to themanufacturer's instructions. Transfected was also varying concentrationsof LNA-antimiRs. After 24 hours, cells were harvested for luciferasemeasurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates was puton an orbital shaker for 30 min. The cells were collected andtransferred to an eppendorf tube and spinned at 10.000 rpm for 30 minafter which 10 μl were transferred to a 96 well plate and luciferasemeasurements were performed according to manufacturer's instructions(Promega).

Example 13 Validation of Interaction of the miR-21 Target Site in thePdcd4-3′-UTR and miR-21 Using the 8-Mer Compound 3205 (SEQ ID NO: 2)LNA-antimiR

The tumour suppressor protein Pdcd4 inhibits TPA-induced neoplastictransformation, tumour promotion and progression. Pdcd4 has also beenshown to be upregulated in apoptosis in response to different inducers.Furthermore, downregulation of Pdcd4 in lung and colorectal cancer hasalso been associated with a poor patient prognosis. Recently, Asanganiet al and Frankel et al showed that the Pdcd4-3′-UTR contains aconserved target site for miR-21, and transfecting cells with anantimiR-21, resulted in an increase in Pdcd4 protein. We thereforeconstructed a luciferase reporter plasmid, harboring 313 nt of the 3′UTRregion of Pdcd4 encompassing the aforementioned miR-21 target site,which was co-transfected together with different LNA-antimiRs into HeLacells. The different LNA-antimiRs were; Compound 3205 (SEQ ID NO:2)(8-mer, perfect match) or Compound 3218 (SEQ ID NO: 16) (8-mer,mismatch). Luciferase measurements were performed after 24 hours.

Results:

As shown in FIG. 12, in cells transfected with the Pdcd4 3′UTRluciferase reporter and Compound 3205 (SEQ ID NO: 2), an increase inluciferase activity was observed, indicating interaction between thePdcd4 3′UTR and miR-21. However, transfecting the cells with themismatch compound, Compound 3218 (SEQ ID NO: 16), no change inluciferase activity was observed, which was expected since the compounddoes not antagonize miR-21. When comparing the 8-mer LNA-antimiR againsttwo longer designed LNA-antimiRs, the short fully LNA-modified andphosphorothiolated LNA-antimiR was significantly more potent, confirmingprevious luciferase assay data.

Conclusion:

These data conclude that Compound 3205 (SEQ ID NO: 2), which antagonizesmiR-21, can regulate the interaction between Pdcd4 3′UTR and miR-21.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

60.000 cells were seeded per well in a 24-well plate the day beforetransfection in order to achieve 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 0.2 ugPdcd4-3′UTR/psiCHECK2 or empty psiCHECK2 vector together with 0.7 μlLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. Varying concentrations of the LNA-antimiR oligonucleotideswere also transfected. After 24 hours, cells were harvested forluciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 100 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates was puton an orbital shaker for 30 min. The cells were collected andtransferred to an eppendorf tube and spinned at 10.000 rpm for 30 minafter which 10 μl were transferred to a 96 well plate and luciferasemeasurements were performed according to manufacturer's instructions(Promega).

Example 14 Comparison of an 8-Mer LNA-antimiR (Compound 3207,SEQ ID NO:4) with a 15-Mer LNA-antimiR (Compound 3206,SEQ ID NO: 3) inAntagonizing miR-155 in Mouse RAW Cells

To ask whether our approach of using short LNA-antimiRs could be adaptedto targeting other miRNAs we designed a fully LNA-modified 8-merLNA-antimiR against microRNA-155. A perfect match target site formiR-155 was cloned into the 3′UTR of the luciferase gene in the reporterplasmid psiCHECK2 and transfected into the mouse RAW macrophage cellline together with an 8-mer or a 15-mer LNA-antimiR. Because theendogenous levels of miR-155 are low in the RAW cell line, the cellswere treated with 100 ng/ml LPS for 24 hours in order to induce miR-155accumulation. After 24 hours, luciferase analysis was performed.

Results:

Luciferase measurements showed that the fully LNA-modified 8-merLNA-antimiR Compound 3207 (SEQ ID NO: 4) targeting miR-155 was similarlyeffective in antagonizing miR-155 compared to the 15-mer LNA-antimiRCompound 3206 (SEQ ID NO: 3) (FIG. 13). Both LNA-antimiRs showed a >50%de-repression of the miR-155 luciferase sensor at 0.25 nM concentrationand inhibited miR-155 in a dose-dependent manner.

Analysis of the miRBase microRNA sequence database showed that themiR-155 recognition sequence, of the LNA-antimiR Compound 3207 (SEQ IDNO: 4) is unique for microRNA-155. However, when decreasing theLNA-antimiR length to 7 nt, it is not specific for only miR-155,mdv1-miR-M4 and kshv-miR-K12-11 (SEQ ID NO: 963) is also targeted.

Conclusion:

A fully LNA-modified and phosphorothiolated 8-mer LNA-antimiR is equallypotent compared with a 15-mer LNA-antimiR of a mixed LNA/DNA design inantagonizing miR-155. Thus, our approach of using short LNA-antimiRs canbe readily adapted to targeting of other miRNAs

Materials and Methods:

Cell Line:

The mouse macrophage RAW 264.7 cell line was purchased from ATCC(TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10%fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

500.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, RAW 264.7 cells were transfected with 0.3 ug miR-155perfect match/psiCHECK2 or empty psiCHECK2 vector together with 10 μlLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. Transfected was also varying concentrations ofLNA-antimiRs. In order to induce miR-155 accumulation, LPS (100 ng/ml)was added to the RAW cells after the 4 hour incubation with thetransfection complexes. After another 24 hours, cells were harvested forluciferase measurements.

Luciferase Assay:

The cells were washed with PBS and harvested with cell scraper, afterwhich cells were spinned for 5 min at 2.500 rpm. The supernatant wasdiscarded and 50 d 1× Passive Lysis Buffer (Promega) was added to thecell pellet, after which cells were put on ice for 30 min. The lysedcells were spinned at 10.000 rpm for 30 min after which 20 μl weretransferred to a 96-well plate and luciferase measurements wereperformed according to the manufacturer's instructions (Promega).

Example 15 Assessment of c/EBPβ Protein Levels as a Functional Readoutfor miR-155 Antagonism by Short LNA-antimiR (Compound 3207, SEQ ID NO:4)

As a functional readout for miR-155 antagonism by short LNA-antimiR(Compound 3207, SEQ ID NO: 4) we determined the protein levels of anovel miR-155 target, c/EBPβ. The mouse macrophage RAW cell line wastransfected together with either an 8-mer (Compound 3207,SEQ ID NO: 4)or a 15-mer (Compound 3206, SEQ ID NO: 3) LNA-antimiR in the absence orpresence of pre-miR-155. As mismatch controls for the 15-mer, Compound 4(SEQ ID NO: 5) was used, which targets miR-122 and for the 8-merCompound 3205 (SEQ ID NO: 2) was used, which targets miR-21. These twocontrol miRNAs do not regulate c/EBPβ expression levels. LPS was used toinduce miR-155 accumulation and cells were harvested after 16 hours withLPS. c/EBPβ has three isoforms; LIP, LAP and LAP* that were detected byWestern blot analysis and the same membranes were re-probed withbeta-tubulin as loading control.

Results:

Ratios were calculated for c/EBPβ LIP and beta-tubulin as indicated inFIG. 14.

RAW cells that were transfected with the 15-mer LNA-antimiR and nopre-miR-155 all showed equal c/EBPβ LIP/beta-tubulin ratios, due toinhibition of miR-155 increases the c/EBPβ LIP levels (FIG. 14, leftpanel). By comparison, transfection of pre-miR-155 in RAW cells resultedin decreased c/EBPβ LIP levels as expected, if c/EBPβ was a miR-155target, as shown in lanes with protein extracts from RAW cells treatedwith no LNA or a mismatch. However, protein extracts from RAW cellstransfected with LNA-antimiR against miR-155, showed an increase ofc/EBPβ LIP levels. The same experiments were also carried out with the8-mer LNA-antimiR-155 (Compound 3207, SEQ ID NO: 4) and as shown in FIG.14 (right panel) comparable results to those with the 15-mer LNA-antimiRCompound 3206 (SEQ ID NO: 3) were obtained.

Conclusion:

Antagonism of miR-155 using either an 8-mer or a 15-mer LNA-antimiRleads to de-repression of the direct target c/EBPβ.

Materials and Methods:

Cell Line:

The mouse macrophage RAW 264.7 cell line was purchased from ATCC(TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10%fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

500.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to achieve 50% confluency the next day. On the dayof transfection, RAW 264.7 cells were transfected with 5 nmolpre-miR-155 (Ambion) and/or 5 nM LNA-antimiR together with 10 μlLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. Transfected was also varying concentrations ofLNA-antimiRs. In order to induce miR-155 accumulation, LPS (100 ng/ml)was added to the RAW cells after the 4 hour incubation with thetransfection complexes. After 16 hours, cells were harvested for proteinextraction and western blot analysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubesand 250 μl lysis buffer (1×RIPA) was added. The cell lysate was placedon ice for 20 min and spinned at 10.000 rpm for 10 minutes. The proteinconcentration was measured with Coomassie Plus according to themanufacturer's instructions and 80 ug was loaded onto a 4-12% BIS-TRISgel. The membrane was incubated overnight at 4° C. with the primarymonoclonal mouse antibody C/EBPβ (Santa Cruz) with a 1:100concentration. Immunoreactive bands were visualized with ECL Plus(Amersham).

Example 16 Antagonism of miR-106b by a Fully LNA-Modified 8-Mer(Compound 3221, SEQ ID NO: 19) LNA-antimiR

To confirm that our approach of using short LNA-antimiRs could beadapted to targeting of other miRNAs we designed a fully LNA-modified8-mer LNA-antimiR against microRNA-106b. A perfect match target site formiR-106b was cloned into the 3′UTR of the luciferase gene in the vector(psiCHECK2) and transfected into the human cervix carcinoma HeLa cellline together with a short LNA-antimiR (Compound 3221, SEQ ID NO: 19) orwith a 15-mer LNA-antimiR (Compound 3228, SEQ ID NO: 26) at varyingconcentrations. Luciferase measurements were performed after 24 hours.

Results:

Transfection of the 8-mer LNA-antimiR Compound 3221 (SEQ ID NO: 19)against miR-106b resulted in dose-dependent inhibition of miR-106b asshown by de-repression of the luciferase reporter, which was completelyde-repressed at 1 nM LNA-antimiR concentration (FIG. 15). Comparableresults were obtained using the 15-mer LNA-antimiR Compound 3228 (SEQ IDNO: 26) demonstrating that an 8-mer LNA-antimiR is similarly potent to a15-mer.

Conclusion:

Targeting of miR-106b in HeLa cells shows that an 8-mer fullyLNA-modified and phosphorothiolated LNA-antimiR is equally potentcompared with a 15-mer LNA/DNA mixmer LNA-antimiR.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

5.200 cells were seeded per well in a 96-well plate the day beforetransfection in order to achieve 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 57 ng miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vectortogether with 0.14 μl Lipofectamine2000 (Invitrogen) according to themanufacturers instructions. Transfected was also varying concentrationsof LNA-antimiRs. After 24 hours, cells were harvested for luciferasemeasurements.

Luciferase Assay:

The cells were washed with PBS and 30 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates was puton an orbital shaker for 30 min. The cells were collected andtransferred to eppendorf tubes and spinned at 10.000 rpm for 30 minafter which luciferase measurements were performed according to themanufacturer's instructions (Promega).

Example 17 Antagonism of miR-19a by a fully LNA-modified 8-mer (Compound3222, SEQ ID NO: 20) LNA-antimiR

To further confirm that our approach of using short LNA-antimiRs can bereadily adapted to targeting of other miRNAs we designed a fullyLNA-modified 8-mer LNA-antimiR against microRNA-19a. A perfect matchtarget site for miR-19a was cloned in the 3′UTR of the luciferase genein the psiCHECK2 vector. The reporter plasmid was transfected into thehuman cervix carcinoma HeLa cell line together with a short LNA-antimiR(Compound 3222, SEQ ID NO: 20) or with a 15-mer LNA-antimiR (Compound3229, SEQ ID NO: 27) targeting miR-19a at varying concentrations.Luciferase measurements were performed after 24 hours.

Results:

As shown in FIG. 16, transfection of the 15-mer LNA-antimiR Compound3229 (SEQ ID NO: 27) into HeLa efficiently antagonizes miR-19a asdemonstrated by complete de-repression at 1 nM LNA-antimiRconcentration. By comparison, transfection of the 8-mer LNA-antimiRCompound 3222 (SEQ ID NO: 20) resulted in effective miR-19a antagonismalready at 0.5 nM concentration, indicating that this 8-mer LNA-antimiRis at least equally potent compared with a 15-mer LNA-antimiR in HeLacells.

Conclusion:

Targeting of miR-19a in HeLa cells shows that an 8-mer fullyLNA-modified and phosphorothiolated LNA-antimiR is at least equallypotent compared with a 15-mer LNA/DNA mixmer LNA-antimiR.

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

5.200 cells were seeded per well in a 96-well plate the day beforetransfection in order to achieve 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 57 ng miR-21perfect match/psiCHECK2, miR-21.mm2/psiCHECK2 or empty psiCHECK2 vectortogether with 0.14 d Lipofectamine2000 (Invitrogen) according tomanufacturer's instructions. Transfected was also varying concentrationsof LNA-antimiRs. After 24 hours, cells were harvested for luciferasemeasurements.

Luciferase Assay:

The cells were washed with PBS and 30 μl 1× Passive Lysis Buffer(Promega) was added to each well, after which the 24-well plates was puton an orbital shaker for 30 min. The cells were collected andtransferred to eppendorf tubes and spinned at 10.000 rpm for 30 minafter which luciferase measurements were performed according to themanufacturer's instructions (Promega).

Example 18 Targeting of a microRNA Family Using Short, FullyLNA-Substituted LNA-antimiR

Next, we investigated whether it is possible to target a microRNA familyusing a single short 7-mer LNA-antimiR complementary to the seedsequence that is common for all family members (see FIG. 17). In thisexperiment, we focused on miR-221 and miR-222 that are overexpressed insolid tumors of the colon, pancreas, prostate and stomach. It has alsobeen shown that miR-221 and miR-222 are the most significantlyupregulated microRNAs in glioblastoma multiforme. Furthermore,overexpression of miR-221 and miR-222 may contribute to the growth andprogression of prostate carcinoma, at least in part by blocking thetumor suppressor protein p27. A perfect match target site for bothmiR-221 and miR-222, respectively, was cloned into the 3′UTR of theluciferase gene resulting in two reporter constructs. These constructswere then transfected either separate or combined into the prostatecarcinoma cell line, PC3. In addition to the 7-mer, targeting bothmiR-221 and miR-222, we also co-transfected a 15-mer LNA-antimiR (15mer) targeting either miR-221 (Compound 3223, SEQ ID NO: 21) or miR-222(Compound 3224, SEQ ID NO: 22), each transfected separately or together(see FIG. 18 left).

Results:

As shown in FIG. 18, transfection of PC3 cells with the LNA-antimiRCompound 3223 (SEQ ID NO: 21) against miR-221 resulted in efficientinhibition of miR-221 at 1 nM LNA-antimiR concentration. An inhibitoryeffect is also observed when using the luciferase reporter plasmid formiR-222 as well as when co-transfecting both luciferase reporters formiR-221 and miR-222 simultaneously into PC3 cells. This inhibitoryeffect is most likely due to the shared seed sequence between miR-221and miR-222. Similarly, transfection of PC3 cells with the LNA-antimiRCompound 3224 (SEQ ID NO: 22) against miR-222 resulted in efficientinhibition of miR-222 at 1 nM LNA-antimiR concentration as shown bycomplete de-repression of the luciferase reporter for miR-222. Aninhibitory effect is also observed when using the luciferase reporterplasmid for miR-222 as well as when co-transfecting both luciferasereporters for miR-221 and miR-222 simultaneously into PC3 cells.Co-transfection of both LNA-antimiR compounds Compound 3223 (SEQ ID NO:21) and Compound 3224 (SEQ ID NO: 22) against miR-221 and miR-222,respectively, (see FIG. 18 left), resulted in effective inhibition ofboth miRNAs as shown by complete de-repression of the luciferasereporter plasmids both when separately transfected and whenco-transfected into PC3 cells. Interestingly, transfection of a singlefully LNA-modified 7-mer LNA-antimiR (Compound 3225, SEQ ID NO: 23)targeting the seed sequence of miR-221 and miR-222 into PC3 cellsresulted in efficient, dose-dependent antagonism of miR-221 and miR-222simultaneously as shown by complete de-repression of the luciferasereporter plasmids both when separately transfected and whenco-transfected into PC3 cells. This demonstrates that a single, shortLNA-substituted LNA-antimiR can effectively target seed sequencesthereby antagonizing entire microRNA families simultaneously. Analysisof the miRBase microRNA sequence database showed that the miR-221/222seed recognition sequence, of the LNA-antimiR Compound 3225 (SEQ ID NO:23) is unique for both miRNAs.

Conclusion:

Our results demonstrate that LNA enables design and synthesis of shortfully LNA-substituted LNA-antimiR oligonucleotides that can effectivelytarget microRNA seed sequences thereby antagonizing entire microRNAfamilies simultaneously.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714) PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

100.000 cells were seeded per well in a 12-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, PC3 cells were transfected with 0.3 ug of luciferasereporter plasmid for miR-221 or for miR-222 or with empty psiCHECK2vector without miRNA target site as control together with 1.2 μlLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. After 24 hours, cells were harvested for luciferasemeasurements.

Luciferase Assay:

The cells were washed with PBS and 250 μl 1× Passive Lysis Buffer(Promega) was added to the wells. The plates were placed on a shaker for30 min., after which the cell lysates was transferred to eppendorftubes. The cell lysate was spinned for 10 min at 2.500 rpm after which20 μl were transferred to a 96-well plate and luciferase measurementswere performed according to the manufacturer's instructions (Promega).

Example 19 Assessment of p27 Protein Levels as a Functional Readout forAntagonism of the miR-221/222 Family by the 7-Mer Compound 3225 (SEQ IDNO: 23) LNA-antimiR

Previous work has shown (le Sage et al. 2007, Galardi et al. 2007) thatmiR-221 and miR-222 post-transcriptionally regulate the expression ofthe tumour suppressor gene p27, which is involved in cell cycleregulation. In these studies, down-regulation of miR-221 and miR-222 wasshown to increase expression levels of p27. Thus, as a functionalreadout for antagonism of the miR-221/222 family by the 7-mer Compound3225 (SEQ ID NO: 23) LNA-antimiR we determined the protein levels of p27after transfection of the LNA-antimiR Compound 3225 (SEQ ID NO: 23) intoPC3 cells in comparison with an 8-mer LNA mismatch control. After 24hours the cells were harvested for western blot analysis (FIG. 19).

Results:

As shown in FIG. 19, transfection of the 7-mer LNA-antimiR Compound 3225(SEQ ID NO: 23) targeting the seed sequence in miR-221 and miR-222resulted in dose-dependent increase of the p27 protein levels comparedto either untransfected or LNA mismatch control transfected PC3 cells.These results clearly demonstrate that the 7-mer LNA-antimiR is able toeffectively antagonize the miR-221/222 family leading to de-repressionof the direct target p27 at the protein level.

Conclusion:

A fully LNA-modified 7-mer LNA-antimiR targeting the seed sequence inthe miR-221/222 family effectively antagonized both miRNAs leading tode-repression of the direct target p27 at the protein level.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714) PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

250.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, PC3 cells were transfected with LNA-antimiRs at varyingconcentrations with Lipofectamine2000. Cells were harvested after 24hours for protein extraction and western blot analysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubesand 250 μl lysis buffer (1×RIPA) was added. The cell lysate was placedon ice for 20 min, then spinned at 10.000 rpm for 10 minutes. Theprotein concentration was measured with Coomassie Plus according to themanufacturer's instructions and 100 ug was loaded onto a 4-12% BIS-TRISgel. The membrane was incubated overnight at 4° C. with the primarymonoclonal mouse antibody p27 (BD Biosciences) at a 1:1000 dilution.Immunoreactive bands were visualized with ECL Plus (Amersham).

Example 20 Duplex Melting Temperatures (T_(m)) of the LNA-antimiRs

As shown in Table 5, T_(m) values increase with increasing the length ofshort fully modified LNA-antimiRs (see T_(m) values for Compound 3205(SEQ ID NO: 2) and Compounds 3209 to 3214 (SEQ ID NOs: 7 to 12) in Table7). Most optimal inhibitory effect was achieved with the 8-merLNA-antimiR Compound 3205 (SEQ ID NO: 2) against miR-21, whereas thevery low Tm of the 6-mer Compound 3209 (SEQ ID NO: 7) is most likely notsufficient to mediate antagonism of the miR-21 target. On the otherhand, increasing the length beyond a 10-mer (Compound 3212, SEQ ID NO:10) significantly increases the T_(m), while simultaneously decreasingthe inhibitory activity as measured using the luciferase miR-21reporter, which is most likely due to high propensity of the fullymodified 12- and 14-mer LNA-antimiRs to form homodimers. The experimentsusing a sliding window of fully LNA-modified 8-mer LNA-antimirs acrossthe mir-21 recognition sequence clearly demonstrate that in addition toadequate T_(m) value of the LNA-antimiR, the seed region is mostcritical for miRNA function and, thus, the most optimal region to betargeted by an LNA-antimiR.

TABLE 5 T_(m) values for miR-21 LNA-antimiRs, measured against acomplementary RNA oligonucleotide SEQ ID Length Measured T_(m) CompoundNO: microRNA (bp) Sequence (RNA) ° C. 3205 2 miR-21 8 5′-GATAAGCT-3′64.0 3209 7 miR-21 6 5′-TAAGCT-3′ 32.0 3210 8 miR-21 7 5′-ATAAGCT-3′45.0 3211 9 miR-21 9 5′-TGATAAGCT-3′ 65.0 3212 10 miR-21 105′-CTGATAAGCT-3′ 63.0 3213 11 miR-21 12 5′-GTCTGATAAGCT-3′ 86.8 3214 12miR-21 14 5′-CAGTCTGATAAGCT-3′ 89.9 3215 13 miR-21 8 5′-TCTGATAA-3′ 56.03216 14 miR-21 8 5′-ATCAGTCT-3 72.0 3217 15 miR-21 8 5′-TCAACATC-3 48.0

Conclusion:

The T_(m) values along with experimental data obtained with luciferasereporters show that potent antagonism by LNA-antimiR is not onlydependent on T, but also depends on the positioning of the LNA-antimiRwithin the microRNA recognition sequence.

Materials and Methods:

T_(m) Measurements:

The oligonucleotide:miR-21 RNA duplexes were diluted to 3 μM in 500 μlRNase free H₂0 and mixed with 500 μl 2×T_(m)-buffer (200 mM NaCl, 0.2 mMEDTA, 20 mM Na-phosphate, pH 7.0). The solution was heated to 95° C. for3 min and then allowed to anneal in RT for 30 min. The duplex meltingtemperatures (T_(m)) were measured on a Lambda 40 UV/VISSpectrophotometer equipped with a Peltier temperature programmer PTP6using PE Templab software (Perkin Elmer). The temperature was ramped upfrom 20° C. to 95° C. and then down to 25° C., recording absorption at260 nm. First derivative and the local maximums of both the melting andannealing were used to assess the duplex melting temperatures.

Example 21 Assessment of miR-21 Antagonism by Comparing an 8-Mer(Compound 3205, SEQ ID NO: 2) Versus a 15-Mer (Compound 3204, SEQ IDNO: 1) LNA-antimiR in Human Hepatocytic Cell Line HepG2

We have previously shown in this application, that an 8-mer LNA-antimiRthat is fully LNA-modified and phosphorothiolated effectivelyantagonizes miR-21 in the human cervix carcinoma cell line HeLa, thehuman breast carcinoma cell line MCF-7 and the human prostate cancercell line PC3. We extended this screening approach to the humanhepatocellular cancer cell line HepG2. To assess the efficiency of the8-mer LNA-antimiR oligonucleotide against miR-21, luciferase reporterconstructs were generated in which a perfect match target site for themature miR-21 was cloned into the 3′UTR of the Renilla luciferase gene.In order to monitor miR-21 inhibition, HepG2 cells were transfected withthe luciferase constructs together with the miR-21 antagonist Compound3205 (SEQ ID NO: 2) (8-mer) and for comparison of specificity with the8-mer LNA-antimiR mismatch (Compound 3218, SEQ ID NO: 16) and forcomparison of potency together with the 15-mer (Compound 3204, SEQ IDNO: 1) at varying concentrations. After 24 hours, luciferase activitywas measured.

Results:

The luciferase reporter experiments showed a dose-dependentde-repression of the luciferase miR-21 reporter activity with the 15-merLNA-antimiR against miR-21 (Compound 3204, SEQ ID NO: 1). However,complete de-repression of the luciferase reporter was not obtained, noteven at the higher concentrations (FIG. 20). In contrast, the cells thatwere transfected with the 8-mer fully LNA modified LNA-antimiR (Compound3205, SEQ ID NO: 2) showed complete de-repression already at 5 nM,indicating significantly improved potency compared to the 15-merLNA-antimiR. Comparing the specificity of the 8-mer perfect match andthe 8-mer mismatch, the mismatch LNA-antimiR (Compound 3218, SEQ ID NO:16) did not show any de-repression at all, demonstrating highspecificity of the LNA-antimiR compound against miR-21.

Conclusion:

The 8-mer (Compound 3205, SEQ ID NO: 2) is more potent than the 15-merLNA-antimiR in targeting miR-21 and antagonism of miR-21 by Compound3205 (SEQ ID NO: 2) is specific.

Materials and Methods:

Cell Line:

The human hepatocytic HepG2 cell line was purchased from ECACC(#85011430). HepG2 cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

650.000 cells were seeded per well in a 6-well plate and reversetransfection were performed. HepG2 cells were transfected with 0.6 μgmiR-21 or empty psiCHECK2 vector together with 2.55 μl Lipofectamine2000(Invitrogen) according to manufacturer's instructions.

Transfected was also varying concentrations of LNA-antimiRs. After 24hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 300 μl 1× Passive Lysis Buffer(Promega) was added to the wells. The plates were placed on a shaker for30 min., after which the cell lysates were transferred to eppendorftubes. The cell lysate was centrifugated for 10 min at 2.500 rpm afterwhich 50 μl were transferred to a 96 well plate and luciferasemeasurements were performed according to the manufacturer's instructions(Promega).

Example 22 Validation of Interaction of the miR-21 Target Site in thePdcd4 3UTR and miR-21 Using the 8-Mer Compound 3205 (SEQ ID NO: 2)LNA-antimiR in Human Hepatocellular Cell Line Huh-7

The tumour suppressor protein Pdcd4 inhibits tumour promotion andprogression. Furthermore, downregulation of Pdcd4 in lung and colorectalcancer has also been associated with poor patient prognosis. Recently,Asangani et al (Oncogene 2007) and Frankel et al (J Biol Chem 2008)showed that the Pdcd4 3′UTR contains a conserved target site for miR-21,and transfecting cells with an antimiR-21, resulted in an increase inPdcd4 protein. We therefore constructed a luciferase reporter plasmid,harboring 313 nt of the 3′UTR region of Pdcd4 encompassing theaforementioned miR-21 target site, which was co-transfected togetherwith different LNA-antimiRs and pre-miR-21 (10 nM) into Huh-7 cells. Thedifferent LNA-antimiRs were; Compound 3205 (SEQ ID NO: 2) (8-mer,perfect match), Compound 3218 (SEQ ID NO: 16) (8-mer, mismatch) andCompound 3204 (SEQ ID NO: 1) (15-mer, DNA/LNA mixmer). Luciferasemeasurements were performed after 24 hours.

Results:

As shown in FIG. 21, cells transfected with the Pdcd4 3′UTR luciferasereporter and Compound 3205 (SEQ ID NO: 2), an increase in luciferaseactivity was observed, indicating interaction between the Pdcd4 3′UTRand miR-21. However, transfecting the cells with the mismatch compound,Compound 3218 (SEQ ID NO: 16), no change in luciferase activity wasobserved, which was expected since the compound does not antagonizemiR-21. When comparing the 8-mer LNA-antimiR against the 15-merLNA-antimiR (Compound 3204. SEQ ID NO: 1), the short fully LNA-modifiedand phosphorothiolated LNA-antimiR was significantly more potent,confirming previous data.

Materials and Methods:

Cell Line:

The human hepatoma cell line Huh-7 was a kind gift from R.Bartinschlager (Dept Mol Virology, University of Heidelberg). Huh-7cells were cultured in DMEM medium, supplemented with 10% fetal bovineserum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

11.000 cells were seeded per well in a 96-well plate the day beforetransfection in order to achieve 50-70% confluency the next day. On theday of transfection, Huh-7 cells were transfected with 20 ng Pdcd43′UTR/psiCHECK2 or empty psiCHECK2 vector together with 10 nM pre-miR-21(Ambion) and 0.14 μl Lipofectamine2000 (Invitrogen) according to themanufacturer's instructions. Varying concentrations of the LNA-antimiRoligonucleotides were also transfected. After 24 hours, cells wereharvested for luciferase measurements.

Luciferase Assay:

Cells were washed and 30 μl 1× Passive Lysis Buffer (Promega) was addedto each well, after which the 96-well plates was put on an orbitalshaker. After 30 min., 50 μl luciferase substrate dissolved inLuciferase Assay Buffer II (Dual-Luciferase Reporter Assay System fromPromega, Cat#E1910) was added to the wells with lysated cells andluciferase measurements were performed according to the manufacturer'sinstructions (Promega).

Example 23 Assessment of Pdcd4 Protein Levels as a Functional Readoutfor miR-21 Antagonism by the 8-Mer LNA-antimiR (Compound 3205, SEQ IDNO: 2)

In addition, we also transfected HeLa cells with Compound 3205 (SEQ IDNO: 2) (perfect match), Compound 3218 (SEQ ID NO: 16) (mismatch),Compound 3219 (SEQ ID NO: 17) (scrambled) and analyzed Pdcd4 proteinlevels after 24 hours with Western blot (FIG. 22). As shown, in theprotein extracts from cells where Compound 3205 (SEQ ID NO: 2) had beenadded, the Pdcd4 protein levels increase, due to antagonism of mir-21 byCompound 3205 (SEQ ID NO: 2) in contrast to the two control LNAoligonucleotides.

Conclusion:

Antagonism of miR-21 using an 8-mer (Compound 3205, SEQ ID NO: 2) leadsto derepression of the direct target Pdcd4 □ntagoism of miR-21

Materials and Methods:

Cell Line:

The human cervix carcinoma cell line HeLa was purchased from ECACC(#93021013). HeLa cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

200.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, HeLa cells were transfected with 5 nM LNAoligonucleotides and 2.5 μg/ml Lipofectamine2000 (Invitrogen) accordingto the manufacturer's instructions. After 24 hours, cells were harvestedfor Western blot analysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubesand 50 μl lysis buffer (1×RIPA) was added. The cell lysate was placed onice for 20 min and spinned at 10.000 rpm for 10 minutes. Equal amounts(15 μl cell lysate) were loaded onto a 4-12% BIS-TRIS gel. The proteinswere transferred to a nitrocellulose membrane using iBlot (Invitrogen)according to manufacturers instructions. The membrane was incubatedovernight at 4° C. with the primary affinity purified rabbit serumantibody Pdcd4 (Rockland) with a 1:2000 concentration. As control,anti-beta tubulin antibodies (Thermo Scientific) were used at a 1:5000dilution. Immunoreactive bands were visualized with ECL Plus (Amersham).

Example 24 Assessment of Potential Hepatotoxicity of the 8-Mer PerfectMatch LNA-antimiR Compound 3205 (SEQ ID NO: 2) and the LNA MismatchControl Compound 3218 (SEQ ID NO: 16)

Each compound was injected into female NMRI mice, at doses of 25 mg/kg,5 mg/kg and 1 mg/kg, every other day for 2 weeks. The animals weresacrificed and serum was collected from whole blood for ALT and ASTanalyses. As seen in FIG. 23, the ALT and AST levels were not elevatedfor Compound 3205 (SEQ ID NO: 2) compared to saline or Compound 3218(SEQ ID NO: 16) (mismatch control). However, one mouse showed increasedlevels (marked red), since the serum samples were contaminated with redblood cells, which contain 6-8 times higher levels of ALT and ASTcompared to plasma. The mice that received 5 mg/kg and 1 mg/kg were alsoanalyzed for ALT and AST levels and showed no changes compared to salinetreated control animals (data not shown).

Materials and Methods: Experimental Design:

Ani- Compound Conc. at Gr. mal No. of Dose level dose vol. Adm. no.IDno. mice per day 10 ml/kg Route Dosing 1  1-10 10 NaCl — i.v 0, 2, 4,7, 9 0.9% 2 11-15 5 Compound 3205 2.5 mg/ml i.v 0, 2, 4, 7, 9 (SEQ IDNO: 2) 25 mg/kg 3 16-20 5 Compound 3205 0.5 mg/ml i.v 0, 2, 4, 7, 9 (SEQID NO: 2) 5 mg/kg 4 21-25 5 Compound 3205 0.1 mg/ml i.v 0, 2, 4, 7, 9(SEQ ID NO: 2) 1 mg/kg 5 26-30 5 Compound 3230 2.5 mg/ml i.v 0, 2, 4, 7,9 (SEQ ID NO: 16) 25 mg/kg 6 31-35 5 Compound 3230 0.5 mg/ml i.v 0, 2,4, 7, 9 (SEQ ID NO: 16) 5 mg/kg

Sacrifice:

The animals was sacrificed by cervical dislocation.

Sampling of Serum for ALT/AST:

The animals were anaesthetised with 70% CO₂-30% O₂ before collection ofretro orbital sinus blood. The blood was collected into S-monovetteSerum-Gel vials. The serum samples were harvested and stored from eachindividual mouse. The blood samples were stored at room temperature fortwo hours and thereafter centrifuged 10 min, 3000 rpm, at room temp. Theserum fractions were harvested into Eppendorf tubes on wet ice.

ALT and AST Measurements;

ALT and AST measurements were performed in 96-well plates using ALT andAST reagents from ABX Pentra (A11A01627—ALT, A11A01629—AST) according tothe manufacturer's instructions. In short, serum samples were diluted2.5 fold with H₂O and each sample was assayed in duplicate. Afteraddition of 50 μl diluted sample or standard (multical from ABXPentra—A11A01652) to each well, 200 μl of 37° C. AST or ALT reagent mixwas added to each well. Kinetic measurements were performed for 5 minwith an interval of 30s at 340 nm and 37° C.

Example 25 Assessment of PU.1 Protein Levels as a Functional Readout formiR-155 Antagonism by Short LNA-antimiR (Compound 3207, SEQ ID NO: 4)

We have previously shown that the 8-mer (Compound 3207, SEQ ID NO: 4)antagonizing miR-155 leads to derepression of the miR-155 targetc/EBPbeta in the mouse macrophage RAW cells. To further verify thepotency of Compound 3207 (SEQ ID NO: 4) we determined the protein levelsof another miR-155 target, PU.1 As a functional readout for miR-155antagonism by short LNA-antimiR (Compound 3207, SEQ ID NO: 4) weperformed Western blot. The antagonism was verified in the humanmonocytic THP-1 cell line which was transfected together with either an8-mer (Compound 3207, SEQ ID NO: 4) perfect match or a 8-mer control LNAin the absence or presence of pre-miR-155. LPS was used to inducemiR-155 accumulation and cells were harvested after 24 hours.

Results:

THP-1 cells that were transfected with pre-miR-155 shows a decrease inPU.1 levels (FIG. 24). Transfecting the cells with the fullyLNA-modified and phosphorothiolated Compound 3207 (SEQ ID NO: 4)effectively antagonizes miR-155, leading to unaltered levels of PU.1protein. By comparison, transfecting the cells with an 8-mer LNAcontrol, PU.1 levels decreased, indicating that antagonism of miR-155 byCompound 3207 (SEQ ID NO: 4) LNA-antimiR is specific.

Conclusion:

Antagonism of miR-155 using an 8-mer leads to de-repression of thedirect target PU.1 in human THP-1 cells.

Materials and Methods:

Cell Line:

The human monocytic THP-1 cell line was purchased from ECACC(#88081201). THP-1 cells were cultured in RPMI with L-glutamine,supplemented with 10% fetal bovine serum.

Transfection:

200.000 cells were seeded per well in a 12-well plate the day before. Onthe day of transfection, THP-1 cells were transfected with 5 nmolpre-miR-155 (Ambion) and/or 5 nM LNA-antimiR together withLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. LPS (100 ng/ml) was added to the cells after the 4 hourincubation with the transfection complexes. After 24 hours, cells wereharvested for protein extraction and western blot analysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubesand 50 μl lysis buffer (1×RIPA) was added. The cell lysate was placed onice for 20 min and spinned at 10.000 rpm for 10 minutes. Equal amounts(15 μl cell lysate) were loaded onto a 4-12% BIS-TRIS gel. The proteinswere transferred to a nitrocellulose membrane using iBlot (Invitrogen)according to manufacturers instructions The membrane was incubatedovernight at 4° C. with the rabbit monoclonal PU.1 antibody (CellSignaling) with a 1:2000 concentration. As equal loading, Tubulin(Thermo Scientific) was used at a 1:5000 dilution. Immunoreactive bandswere visualized with ECL Plus (Amersham).

Example 26 Assessment of p27 Protein Levels as a Functional Readout forAntagonism of the miR-221/222 Family by the 7-Mer Compound 3225 (SEQ IDNO: 23) LNA-antimiR

Previous work has shown (le Sage et al. 2007, Galardi et al. 2007) thatmiR-221 and miR-222 post-transcriptionally regulate the expression ofthe tumour suppressor gene p27, which is involved in cell cycleregulation. In these studies, down-regulation of miR-221 and miR-222 wasshown to increase expression levels of p27. Thus, as a functionalreadout for antagonism of the miR-221/222 family by the 7-mer Compound3225 (SEQ ID NO: 23) LNA-antimiR we determined the protein levels of p27after transfection of the LNA-antimiR Compound 3225 (SEQ ID NO: 23) intoPC3 cells.

Results:

As shown in FIG. 25, transfection of the 7-mer LNA-antimiR Compound 3225(SEQ ID NO: 23) targeting the seed sequence of miR-221 and miR-222resulted in dose-dependent increase of the p27 protein levels comparedto either untransfected or our LNA scrambled control transfected PC3cells. These results clearly demonstrate that the 7-mer LNA-antimiR isable to effectively antagonize the miR-221/222 family leading tode-repression of the direct target p27 at the protein level atconcentrations as low as 5 nM.

Conclusion:

A fully LNA-modified 7-mer LNA-antimiR targeting the seed sequence inthe miR-221/222 family at 5 nM can effectively antagonize both miRNAsleading to de-repression of the direct target p27 at protein level.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714). PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

250.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, PC3 cells were transfected with LNA-oligonucleotides atvarying concentrations (see FIG. 25) with Lipofectamine2000. Cells wereharvested after 24 hours for protein extraction and western blotanalysis.

Western Blot:

Cells were washed with PBS, trypsinated, transferred to eppendorf tubesand 50 μl lysis buffer (1×RIPA) was added. The cell lysate was placed onice for 20 min, then spinned at 10.000 rpm for 10 minutes. Equal amounts(15 μl cell lysate) were loaded onto a 4-12% BIS-TRIS gel. The proteinswere transferred to a nitrocellulose membrane using iBlot (Invitrogen)according to manufacturers instructions. The membrane was incubatedovernight at 4° C. with the primary monoclonal mouse antibody p27 (BDBiosciences) at a 1:1000 dilution. As loading control, Tubulin (ThermoScientific) was used at a 1:5000 dilution. Immunoreactive bands werevisualized with ECL Plus (Amersham).

Example 27 Knock-Down of miR-221/222 by the 7-Mer Compound 3225 (SEQ IDNO: 23) LNA-antimiR Reduces Colony Formation of PC3 Cells

A hallmark of cellular transformation is the ability for tumour cells togrow in an anchorage-independent way in semisolid medium. We havetherefore performed soft agar assay which is a phenotypic assay that isrelevant for cancer, given that it measures the decrease of tumourcells. We transfected Compound 3225 (SEQ ID NO: 23) (perfect match) andCompound 3231 (SEQ ID NO: 28) (scrambled) into PC3 cells, and after 24hours plated cells in soft agar. Colonies were counted after 12 days. Weshow in FIG. 26 that inhibition of miR-221 and miR-222 by Compound 3225(SEQ ID NO: 23) can reduce the amount of colonies growing in soft agarcompared to the scrambled control LNA-antimiR, indicating decrease oftumour cells.

Conclusion:

The 7-mer (Compound 3225, SEQ ID NO: 23) targeting the miR-221/222family reduces the number of colonies in soft agar, indicatingproliferation arrest of PC3 cells.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714). PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

250.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, PC3 cells were transfected with 25 nM of different LNAoligonucleotides with Lipofectamine2000.

Clonogenic Growth in Soft Agar:

2.5×10³ PC3 cells were seeded in 0.35% agar on the top of a base layercontaining 0.5% agar. Cells were plated 24 hours after transfection.Plates were incubated in at 37° C., 5% CO₂ in a humified incubator for12 days and stained with 0.005% crystal violet for 1 h, after whichcells were counted. The assay was performed in triplicate.

Example 28 Assessment of Let-7 Antagonism by 6-9-Mer LNA-antimiRs inHuh-7 Cells Transfected with Let-7a Precursor miRNA, and a LuciferaseSensor Assay

In order to assess the efficiency of fully LNA-modified 6-9-meroligonucleotides in targeting and antagonizing the let-7 family ofmiRNAs, a luciferase sensor construct was made, containing some 800 bpof the HMGA2 3′UTR. The sequence cloned into the vector contains fourout of seven functional let-7 binding sites (sites 2-5), as previouslydemonstrated by Mayr et al. (Science, 2007) and Lee and Dutta (GenesDev, 2007). In order to monitor let-7 inhibition, the hepatocellularcarcinoma cell line Huh-7 (with low to non-existing levels of endogenouslet-7) was transfected with the luciferase sensor construct, with let-7aprecursor miRNA, and with the 6-9 mer let-7 antagonists Compound 3232(SEQ ID NO: 29), Compound 3233 (SEQ ID NO: 30), Compound 3227 (SEQ IDNO: 25), Compound 3234 (SEQ ID NO: 31), Compound 3235 (SEQ ID NO: 32);see FIG. 27) at increasing concentrations. The 6-9-mer LNA-antimiRs werecompared with Compound 3226 (SEQ ID NO: 33), a 15-mer against let-7a asa positive control. After 24 hours, luciferase activity was measured.

Results:

As seen in FIG. 28, the fully LNA-modified 8- and 9-mer LNA-antimiRs(Compound 3227 (SEQ ID NO: 34), Compound 3234 (SEQ ID NO: 31), andCompound 3235 (SEQ ID NO: 32)) show similar potencies in de-repressingthe let-7 targets in the luciferase sensor assay, as the positivecontrol 15-mer Compound 3226 (SEQ ID NO: 24). Full target de-repressionfor these highly potent compounds is achieved already at 1-5 nM, whereasthe 7-mer Compound 3233 (SEQ ID NO: 30) needs to be present at slightlyhigher concentrations (10 nM) to generate the same effect. However, the6-mer Compound 3232 (SEQ ID NO: 29) shows no effect even at as highconcentrations as 50 nM. The de-repression of luciferase activity by the7-9- and the 15-mer LNA-antimiRs is dose-dependent, which isparticularly clear in the case of the slightly less potent Compound 3233(SEQ ID NO: 30).

Conclusion:

To conclude, the 8-9-mer LNA-antimiRs (Compound 3227 (SEQ ID NO: 25),Compound 3234 (SEQ ID NO: 31), and Compound 3235 (SEQ ID NO: 32)) showequal antagonist potencies in inhibition of let-7a in vitro compared tothe 15-mer LNA-antimiR Compound 3226 (SEQ ID NO: 24) targeting let-7a. Apotent effect, albeit at slightly higher concentrations is also seen forthe 7-mer Compound 3233 (SEQ ID NO: 30), whereas a 6-mer has no effectat tested concentrations.

Materials and Methods:

Cell Line:

The hepatocellular carcinoma cell line Huh-7 was a kind gift from R.Bartinschlager (Dept Mol Virology. University of Heidelberg).Huh-7 cellswere cultured in DMEM medium, supplemented with 10% fetal bovine serum,2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

8,000 cells were seeded per well in a 96-well plate the day beforetransfection in order to receive 60-80% confluency the next day. On theday of transfection, Huh-7 cells in each well were transfected with 20ng HMGA2 3′UTR/psiCHECK2 plasmid, let-7a precursor miRNA (Dharmacon; 10nM end-concentration), LNA-antimiRs Compound 3232 (SEQ ID NO: 29),Compound 3233 (SEQ ID NO: 30), Compound 3227 (SEQ ID NO: 25). Compound3234 (SEQ ID NO: 31), Compound 3235 (SEQ ID NO: 32), Compound 3226 (SEQID NO: 24); 0-50 nM end concentrations) together with 0.17 dLipofectamine2000 (Invitrogen) according to manufacturer's instructions.After 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

Growth media was discarded and 30 μl 1× Passive Lysis Buffer (Promega)was added to each well. After 15-30 minutes of incubation on an orbitalshaker, renilla and firefly luciferase measurements were performedaccording to manufacturer's instructions (Promega).

Example 29 Assessment of Entire Let-7 Family Antagonism by 8-, and15-Mer LNA-antimiRs in Huh-7 Cells Transfected with a Luciferase SensorAssay

In order to assess the efficiency of a fully LNA-modified 8-meroligonucleotide in antagonizing the entire let-7 family of miRNAs, thesame luciferase sensor construct as described in the previous examplewas used. Again, Huh-7 cells (with low to non-existing levels ofendogenous let-7) were transfected with the sensor construct, with oneof the family-representative let-7a, let-7d, let-7e, or let-7iprecursors, and with the antagonist Compound 3227 (SEQ ID NO: 25) atincreasing concentrations. The 8-mer LNA-antimiR was compared toCompound 3226 (SEQ ID NO: 24), a 15-mer against let-7a as a positive andpotent control. After 24 hours, luciferase activity was measured.

Results:

As seen in FIG. 29 the fully LNA-modified 8-mer LNA-antimiRs (Compound3227) (SEQ ID NO: 25) show similar potencies in de-repressing thevarious let-7 targets in the luciferase sensor assay, as the positivecontrol 15-mer Compound 3226 (SEQ ID NO: 24). Nearly full targetde-repression for the 8-mer is achieved already at 0.5-1 nM, except inthe case with let-7e premiR (FIG. 29C), to which only 7 out of 8nucleotides of Compound 3227 (SEQ ID NO: 25) hybridizes to the target.However, despite the terminal mismatch in this case, Compound 3227 (SEQID NO: 25) generates full target de-repression at 5 nM. The positivecontrol 15-mer shows potent antagonism of all precursors and givesnearly full de-repression at 0.5 nM. The de-repression of luciferaseactivity by both the 8- and the 15-mer LNA-antimiRs is clearlydose-dependent, as seen in all four panels (FIG. 29A-D).

Conclusion:

To conclude, the 8-mer LNA-antimiR (Compound 3227, SEQ ID NO: 25), is apotent antagonist against four representative let-7 family members invitro, and thus likely against the entire family. Compared to a 15-merpositive control antagonist, Compound 3226 (SEQ ID NO: 24), the 8-mer isequally potent for three of four targets, and slightly less potent forthe fourth target, let-7e, explained by a terminal mismatch in thiscase.

Materials and Methods:

Cell Line:

The hepatocellular carcinoma cell line Huh-7 was a kind gift from R.Bartinschlager (Dept Mol Virology, University of Heidelberg). Huh-7cells were cultured in DMEM medium, supplemented with 10% fetal bovineserum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

8,000 cells were seeded per well in a 96-well plate the day beforetransfection in order to receive 60-80% confluency the next day. On theday of transfection, Huh-7 cells in each well were transfected with 20ng HMGA2 3′UTR/psiCHECK2 plasmid, with let-7a, -7d, -7e, or -7iprecursor miRNA (Dharmacon: 10 nM end-concentration), and withLNA-antimiRs Compound 3227 (SEQ ID NO: 25) and Compound 3226 (SEQ ID NO:24); 0-50 nM end concentrations) together with 0.17 μl Lipofectamine2000(Invitrogen) according to manufacturer's instructions. After 24 hours,cells were harvested for luciferase measurements.

Luciferase Assay:

Growth medium was discarded and 30 d 1× Passive Lysis Buffer (Promega)was added to each well. After 15-30 minutes of incubation on an orbitalshaker, renilla and firefly luciferase measurements were performedaccording to manufacturer's instructions (Promega).

Example 30 Assessment of Endogenous Let-7 Antagonism by Compound 3227(SEQ ID NO: 25), an 8-Mer LNA-antimiRs, in HeLa Cells Transfected with aLuciferase Sensor Assay

In order to determine the efficiency of a fully LNA-modified 8-meroligonucleotide in targeting and antagonizing endogenous let-7, the sameluciferase sensor construct as described in previous two examples, wasco-transfected with Compound 3227 (SEQ ID NO: 25) into the cervicalcancer cell line HeLa (that expresses moderate to high levels of let-7as determined by Q-PCR; data not shown). Empty psiCHECK-2 vector wasincluded as a negative control.

Results:

As seen in FIG. 30, the fully LNA-modified 8-mer LNA-antimiR Compound3227 (SEQ ID NO: 25) shows potent antagonism of endogenous let-7, andgives full target de-repression at concentrations of 5-10 nM. Thede-repression of luciferase activity is dose-dependent, starting around1 nM and reaching a plateau at approximately 10 nM.

Conclusion:

To conclude, the 8-mer LNA-antimiR (Compound 3227, SEQ ID NO: 25), is apotent antagonist against also endogenous let-7 in vitro, and thusprovides definite evidence that entire miRNA families can besuccessfully targeted by short and fully LNA-modified antagonists.

Materials and Methods:

Cell Line:

The cervical cancer cell line HeLa was purchased from ATCC (#CCL-2™).HeLa cells were cultured in Eagle's MEM medium, supplemented with 10%fetal bovine serum, 2 mM Glutamax, 1×NEAA and 25 ug/ml Gentamicin.

Transfection:

8,000 cells were seeded per well in a 96-well plate the day beforetransfection in order to receive 50-70% confluency the next day. On theday of transfection, HeLa cells in each well were co-transfected with 20ng HMGA2 3′UTR/psiCHECK2 plasmid or psiCHECK-2 (empty vector), and withLNA-antimiR Compound 3227 (SEQ ID NO: 25) (0-50 nM, end concentrations)together with 0.17 μl Lipofectamine2000 (Invitrogen) according tomanufacturer's instructions. After 24 hours, cells were harvested forluciferase measurements.

Luciferase Assay:

Growth media was discarded and 30 μl 1× Passive Lysis Buffer (Promega)was added to each well. After 15-30 minutes of incubation on an orbitalshaker, renilla and firefly luciferase measurements were performedaccording to manufacturer's instructions (Promega).

Example 31 Assessment of miR-21 Antagonism by an 8-Mer LNA-antimiR-21(Compound 3205, SEQ ID NO: 2) Versus an 8-Mer (Compound 3219, SEQ ID NO:17) Scrambled Control LNA in the Human Colon Carcinoma Cell Line HCT116

We have previously shown in this application, that an 8-mer LNA-antimiRthat is fully LNA-modified and phosphorothiolated effectivelyantagonizes miR-21 in the human cervix carcinoma cell line HeLa, thehuman breast carcinoma cell line MCF-7, the human prostate cancer cellline PC3 and human hepatocellular carcinoma HepG2 cell line. We extendedthis screening approach to the human colon carcinoma cell line HCT116.To assess the efficiency of the 8-mer LNA-antimiR oligonucleotideagainst miR-21, luciferase reporter constructs were generated in which aperfect match target site for the mature miR-21 was cloned into the3′UTR of the Renilla luciferase gene. In order to monitor miR-21inhibition, HCT116 cells were transfected with the luciferase constructstogether with the miR-21 antagonist Compound 3205 (SEQ ID NO: 2) (8-mer)and for comparison of specificity with the 8-mer LNA scrambled control(Compound 3219, SEQ ID NO: 17). After 24 hours, luciferase activity wasmeasured.

Results:

The luciferase reporter experiments showed a dose-dependentde-repression of the luciferase miR-21 reporter activity with the 8-merLNA-antimiR against miR-21 (Compound 3205, SEQ ID NO: 2) and completede-repression was obtained at 5 nM (FIG. 31). When comparing thespecificity of the 8-mer perfect match and the 8-mer scrambled control,the scrambled control LNA-antimiR (Compound 3219, SEQ ID NO: 17) did notshow any de-repression at all, demonstrating high specificity of theLNA-antimiR compound against miR-21.

Conclusion:

The 8-mer (Compound 3205, SEQ ID NO: 2) is potent in targeting miR-21and antagonism of miR-21 by Compound 3205 (SEQ ID NO: 2) is specific.

Materials and Methods:

Cell Line:

The human colon carcinoma HCT116 cell line was purchased from ATCC(CCL-247). HCT116 cells were cultured in RPMI medium, supplemented with10% fetal bovine serum, and 25 ug/ml Gentamicin.

Transfection:

110.000 cells were seeded per well in a 12-well plate and transfectionwas performed. HCT116 cells were transfected with 0.3 μg miR-21luciferase sensor plasmid or empty psiCHECK2 vector together with 1.2 μlLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. Transfected were also varying concentrations ofLNA-antimiR and control oligonucleotides. After 24 hours, cells wereharvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and 250 μl 1× Passive Lysis Buffer(Promega) was added to the wells. The plates were placed on a shaker for30 min., after which the cell lysates were transferred to eppendorftubes. The cell lysate was centrifugated for 10 min at 2.500 rpm afterwhich 50 μl were transferred to a 96 well plate and luciferasemeasurements were performed according to the manufacturer's instructions(Promega).

Example 32 Knock-Down of miR-21 by the 8-Mer Compound 3205 (SEQ ID NO:2) LNA-antimiR Reduces Colony Formation of PC3 Cells

A hallmark of cellular transformation is the ability for tumour cells togrow in an anchorage-independent way in semisolid medium. We thereforeperformed soft agar assay which is a phenotypic assay that is relevantfor cancer, given that it measures the decrease of tumour cells. Wetransfected Compound 3205 (SEQ ID NO: 2) (perfect match LNA-antimiR-21)and Compound 3219 (SEQ ID NO: 17) (LNA scrambled control) into PC3cells, and after 24 hours plated cells in soft agar. Colonies werecounted after 12 days. We show in FIG. 32 that inhibition of miR-21 byCompound 3205 (SEQ ID NO: 2) can reduce the amount of colonies growingin soft agar compared to the scrambled control LNA treated or untreatedcontrol (transfected, but with no LNA), demonstrating decrease of tumourcells.

Conclusion:

The 8-mer (Compound 3205, SEQ ID NO: 2) targeting the miR-21 familyreduces the number of colonies in soft agar, demonstrating proliferationarrest of PC3 cells.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714). PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

250.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, PC3 cells were transfected with 25 nM of different LNAoligonucleotides with Lipofectamine2000.

Clonogenic Growth in Soft Agar:

2.5×10³ PC3 cells were seeded in 0.35% agar on the top of a base layercontaining 0.5% agar. Cells were plated 24 hours after transfection.Plates were incubated in at 37° C., 5% CO₂ in a humified incubator for12 days and stained with 0.005% crystal violet for 1 h, after whichcells were counted. The assay was performed in triplicate.

Example 33 Silencing of miR-21 by the 8-Mer Compound 3205 (SEQ ID NO: 2)LNA-antimiR Reduces Colony Formation of HepG2 Cells

miR-21 is overexpressed in the human hepatocellular carcinoma cell lineHepG2 and we have previously shown that we are able to regulate theluciferase activity of a miR-21 sensor plasmid with Compound 3205 (SEQID NO: 2) in these cells. HepG2 cells were transfected with Compound3205 (SEQ ID NO: 2) and Compound 3219 (SEQ ID NO: 17) (scrambled 8-mer),and after 24 hours plated into soft agar. Colonies were counted after 17days with a microscope.

Results:

We show in FIG. 33 that inhibition of miR-21 by Compound 3205 (SEQ IDNO: 2) can reduce the amount of colonies growing in soft agar, showingthat proliferation arrest has occurred. In addition, our scrambled 8-mercontrol, Compound 3219 (SEQ ID NO: 17), had no significant effect on thenumber of colonies.

Conclusion:

The 8-mer (Compound 3205, SEQ ID NO: 2) targeting the miR-21 reduces thenumber of colonies in soft agar, indicating proliferation arrest ofHepG2 cells.

Materials and Methods:

Cell Line:

The human hepatocytic HepG2 cell line was purchased from ECACC(#85011430). HepG2 cells were cultured in EMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

650.000 cells were seeded per well in a 6-well plate and reversetransfection was performed. HepG2 cells were transfected with 0.6 gmiR-21 luciferase sensor plasmid or empty psiCHECK2 vector together with2.55 μl Lipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions. Transfected were also LNA-antimiR and controloligonucleotides as varying concentrations. After 24 hours, the cellswere harvested for luciferase measurements.

Clonogenic Growth in Soft Agar:

2.0×10³ HepG2 cells were seeded in 0.35% agar on the top of a base layercontaining 0.5% agar. Cells were plated 24 hours after transfection.Plates were incubated in at 37° C., 5% CO₂ in a humified incubator for17 days and stained with 0.005% crystal violet for 1 h, after whichcells were counted. The assay was performed in triplicate.

Example 34 Silencing of miR-21 by the 8-Mer Compound 3205 (SEQ ID NO: 2)LNA-antimiR Inhibits Cell Migration in PC3 Cells

Cell migration can be monitored by performing a wound healing assay(=scratch assay) where a “scratch” is made in a cell monolayer, andimages are captured at the beginning and at regular intervals duringcell migration. By comparing the images, quantification of the migrationrate of the cells can be determined. This was done in the human prostatecancer cell line PC3. Cells were seeded, and on day 3 the cells weretransfected, and the next day, when 100% confluency was reached, ascratch (=wound) was made. When the scratch was made, pictures weretaken in order to document the initial wound. Afterwards the area of thewound closure is measured at different time points with the freesoftware program Image J. As shown in FIG. 34A, PC3 cells had beentreated with 25 nM Compound 3205 (SEQ ID NO: 2) (perfect match, miR-21),the control Compound 3219 (SEQ ID NO: 17) or left untransfected.Pictures were taken after 24 hours, and the area was calculated for thewound closure at respective time-point. The wound closure for theuntransfected cells and for the control. Compound 3219 (SEQ ID NO: 17),was faster as compared to our LNA-antimiR against miR-21, Compound 3205(SEQ ID NO: 2), indicating that Compound 3205 (SEQ ID NO: 2) inhibitsmiR-21 and prevents the cells from migrating (FIG. 34B).

Conclusion:

The 8-mer (Compound 3205, SEQ ID NO: 2) targeting miR-21 inhibits thecell migration of PC3 cells compared to untransfected and controltransfected cells.

Materials and Methods:

Cell Line:

The human prostate carcinoma PC3 cell line was purchased from ECACC(#90112714). PC3 cells were cultured in DMEM medium, supplemented with10% fetal bovine serum, 2 mM Glutamax and 25 ug/ml Gentamicin.

Scratch assay: 150.000 cells were seeded per well in a 6-well platethree days before transfection in order to receive 100% confluency thenext day. At 24 hours after transfection, a scratch was made in the cellmonolayer with a 200 μl tip. Pictures were taken at 0 h and after 24hours by using a digital camera coupled to a microscope. The softwareprogram Image J was used to determine wound closure.

Example 35 Length Assessment of Fully LNA-Substituted LNA-antimiRsAntagonizing miR-155

We have previously shown a length assessment for miR-21 regarding fullyLNA-substituted LNA-antimiRs, and showed that the most potentLNA-antimiRs are 7-, 8- or 9 nt in length. The same experiment wasrepeated with miR-155. A perfect match target site for miR-155 wascloned into the 3′UTR of the luciferase gene in the reporter plasmidpsiCHECK2 and transfected into the mouse RAW macrophage cell linetogether with fully LNA-substituted LNA-antimiRs of different lengths.Because the endogenous levels of miR-155 are low in the RAW cell line,the cells were treated with 100 ng/ml LPS for 24 hours in order toinduce miR-155 accumulation. After 24 hours, luciferase analysis wasperformed.

Results:

As shown in FIG. 35, the most potent LNA-antimiRs are Compound 3207 (SEQID NO: 4) (8 nt) and Compound 3241 (SEQ ID NO: 38) (9 nt), reachingalmost a 80% de-repression at only 0.25 nM LNA concentration. The 6-mer(Compound 3244, SEQ ID NO: 978) shows no significant de-repression.Increasing the length to 12-mer to 14-mer (Compound 3242 (SEQ ID NO: 39)and Compound 3243 (SEQ ID NO: 977)) decreased the potency as shown byless efficient de-repression of the miR-155 reporter.

Conclusion:

The most potent fully LNA-substituted LNA-antimiRs targeting miR-155were an 8- and 9-mer (Compound 3207 (SEQ ID NO: 4) and Compound 3241(SEQ ID NO: 38)).

Materials and Methods:

Cell Line:

The mouse macrophage RAW 264.7 cell line was purchased from ATCC(TIB-71). RAW cells were cultured in DMEM medium, supplemented with 10%fetal bovine serum, 4 mM Glutamax and 25 ug/ml Gentamicin.

Transfection:

500.000 cells were seeded per well in a 6-well plate the day beforetransfection in order to receive 50% confluency the next day. On the dayof transfection, RAW 264.7 cells were transfected with 0.3 ug miR-155perfect match/psiCHECK2 or empty psiCHECK2 vector together with 10 μlLipofectamine2000 (Invitrogen) according to the manufacturer'sinstructions.

Transfected was also varying concentrations of LNA-antimiRs. In order toinduce miR-155 accumulation, LPS (100 ng/ml) was added to the RAW cellsafter the 4 hour incubation with the transfection complexes. Afteranother 24 hours, cells were harvested for luciferase measurements.

Luciferase Assay:

The cells were washed with PBS and harvested with cell scraper, afterwhich cells were spinned for 5 min at 2.500 rpm. The supernatant wasdiscarded and 50 μl 1× Passive Lysis Buffer (Promega) was added to thecell pellet, after which cells were put on ice for 30 min. The lysedcells were spinned at 10.000 rpm for 30 min after which 20 μl weretransferred to a 96-well plate and luciferase measurements wereperformed according to the manufacturer's instructions (Promega).

Example 36 Plasma Protein Binding for the Fully LNA-Substituted 8-MerCompound 3205 (SEQ ID NO: 2) Targeting miR-21 (LNA-antimiR-21)

The plasma proteins are not saturated with Compound 3205 (SEQ ID NO:2)at the plasma concentrations in the experiment shown in FIG. 36A. In awide range of Compound 3205 (SEQ ID NO: 2) concentrations in the plasmathe protein binding is around 95% of the Compound 3205 (SEQ ID NO: 2)LNA-antimiR-21 in FIG. 36B. At Compound 3205 (SEQ ID NO: 2)concentrations 50.1 μM (174 μg/mL) the binding capacity of plasmaproteins for FAM-labeled Compound 3205 (SEQ ID NO: 2) has not beensaturated.

Materials and Methods:

Mouse plasma (100 μL) was spiked with FAM-labeled Compound 3205 (SEQ IDNO: 2) to 0.167, 1.67, 5.01, 10.02, 16.7, 25.05 and 50.1 μMconcentrations. The solutions were incubated at 37° C. for 30 minutes.The solutions were transferred to a Microcon Ultracel YM-30 filter(regenerated cellulose 30.000 MWCO). The filters were spun for 20minutes at 2000 g and at room temperature in a microcentrifuge. Thefiltrate was diluted 5, 10 and 20 times and 100 μL samples weretransferred to a microtiter plate (Polystyrene Black NUNC-237108). Thefluorescence was detected using a FLUOstar Optima elisa reader withexcitation 458 nm and emission 520 nm. The amount of unbound FAM-labeledCompound 3205 (SEQ ID NO: 2) was calculated from a standard curvederived from filtrated plasma spiked with FAM-labeled Compound 3205 (SEQID NO: 2) at 12 different (0.45-1000 nM) concentrations. The numberswere corrected with the recovery number established from filtrationexperiments with Compound 3205 (SEQ ID NO: 2) concentrations 0.167,1.67, 5.01, 10.02, 16.7, 25.05 and 50.1 μM in filtrated plasma. Therecovery of FAM-labeled Compound 3205 (SEQ ID NO: 2) was 86%.

Example 37 Quantitative Whole Body Autoradiography Study in FemalePigmented Mice after Single Intravenous Administration of ³⁵S-LabelledCompound 3205 (SEQ ID NO: 2) LNA-antimiR-21

In order to determine the biodistribution of a short fully LNA-modifiedLNA-antimiR (Compound 3205 (SEQ ID NO: 2), 8-mer) a whole body tissuedistribution of radioactively labeled compound was done in mice.MS-labelled Compound 3205 (SEQ ID NO: 2) was dosed to mice with a singleintravenous administration and mice were sacrificed at differenttime-points, ranging from 5 min to 21 days.

TABLE 6(i) Individual tissue concentrations (μg Compound 3205/g tissue)after a single intravenous administration of ³⁵S-labelled Compound 3205(SEQ ID NO: 2) in female pigmented mice. The figures are mean values ofthree measurements for each tissue and ratio. The coefficient ofvariation (CV) is generally about 10%. Max. Conc. of oligo μg CompoundTime of max Tissue 3205/g tissue conc. hours T½ hours Adrenal gl. 13.60.083 374 Bile 4 1 Bone marrow 7.2 0.083 411 Brain 0.4 0.083 Brown fat8.8 0.083 Gastric muc. 10.1 0.083 Heart blood 26.2 0.083 10.3 Kidneyctx. 58.7 24 104 Liver 11.8 0.083 538 10.7 24 Lung 13.2 0.083 239 Lymphnode 5 0.083 262 2.4 48 Lymph 18.8 4 20.8 168 Myocardium 8.1 0.083 662Ovary 13 0.083 198 Pancreas 5 0.083 Pituitary gl. 6.7 0.083 Salivary gl.8.6 0.083 405 5.5 168 skel. Muscle 4.8 0.083 Skin pig. 5.4 0.25 Spleen9.8 0.083 564 Thymus 3.8 0.083 185 Thyroid gl. 10.9 0.083 592 Urine328.9 0.083 Uterus 9.6 0.25 177 Uvea of the eye 13.6 0.083 LOQ 0.0450.083 0.033 24 0.03 168

TABLE 6(ii) Tissue to liver ratios after single intravenousadministration of ³⁵S- labelled Compound 3205 (SEQ ID NO: 2) in femalepigmented mice. ³⁵S-Compound 3205 (SEQ ID NO: 2) Animal no 10 11 12 1314 15 16 17 18 Surv. Time (h) 0.083 0.25 1 h 4 h 24 h 48 h 96 h 168 504Organ Adrenal gl liver liver liver liver liver liver liver liver liverBile 1.15 1.08 0.52 0.27 0.24 0.26 0.23 0.18 0.17 Bone marrow 0.03 0.110.55 0.10 0.03 0.07 0.04 0.03 0.04 Brain 0.61 0.81 0.55 0.45 0.40 0.480.43 0.42 0.34 Brown fat 0.03 0.03 0.01 0.00 0.00 0.00 0.00 0.00 0.00Gastric muc 0.75 0.57 0.29 0.12 0.07 0.12 0.08 0.10 0.07 Heart blood0.86 0.71 0.31 0.22 0.10 0.21 0.15 0.16 0.12 Kidney ctx 2.23 1.91 0.740.11 0.01 0.00 0.00 0.00 0.00 Liver 2.87 3.94 6.45 6.95 5.51 6.68 3.922.24 0.40 Lung 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 Lymph node1.12 0.97 0.63 0.09 0.04 0.04 0.03 0.02 0.02 Lymph 0.43 0.30 0.25 0.190.11 0.32 0.20 0.17 0.12 Myocardium 0.82 1.09 1.78 2.78 1.03 2.05 1.623.17 1.89 Ovary 0.69 0.63 0.30 0.13 0.10 0.15 0.09 0.11 0.12 Pancreas1.10 1.40 0.61 0.31 0.27 0.28 0.21 0.21 0.08 Pituitary gland 0.42 0.370.22 0.18 0.12 0.17 0.12 0.15 0.11 Salivary gland 0.57 0.54 0.28 0.110.15 0.16 0.12 0.10 0.08 Skel. muscle 0.73 0.81 0.38 0.25 0.25 0.42 0.230.85 0.24 Skin, pigm. 0.40 0.28 0.14 0.04 0.02 0.04 0.03 0.03 0.03Spleen 0.34 0.69 0.65 0.36 0.20 0.26 0.20 0.19 0.13 Thymus 0.83 0.860.44 0.32 0.24 0.34 0.35 0.29 0.31 Thyroid gland 0.32 0.31 0.14 0.070.09 0.08 0.05 0.04 0.02 Urine 0.9 1.2 0.43 0.28 0.25 0.34 0.19 0.260.25 Uterus 27.96 39.48 9.90 5.44 0.24 0.39 0.12 0.15 0.03 Uvea of theeye 0.56 1.23 0.65 0.30 0.30 0.07 0.27 0.16 0.08

Conclusions:

Compound 3205 (SEQ ID NO: 2) shows blood clearance of radioactivity withelimination half-lives of 8-10 hours. High levels of radioactivity wereregistered in the kidney cortex, lymph, liver, bone marrow, spleen,ovary and uterus. The highest level of radioactivity was registered inthe kidney cortex showing five times higher levels than that of theliver for Compound 3205 (SEQ ID NO: 2). A strong retention ofradioactivity was noticed in the kidney cortex, lymph, liver, bonemarrow and spleen for Compound 3205 (SEQ ID NO: 2) LNA-antimiR-21.

Materials and Methods:

Dose Administration:

All mice were weighed before administration. Nine female mice were given10 mg/kg of 3S-Compound 3205 (SEQ ID NO: 2) intravenously in a tailvein. The volume given to each animal was 10 mL/kg of the testformulation. The specific activity 75.7 μCi/mg. Individual mice werekilled 5 min, 15 min, 1 hour, 4 hours, 24 hours, 2 days, 4 days, 7 daysand 21 days after administration of Compound 3205 (SEQ ID NO: 2). Wholebody autoradiography: The mice were anaesthetized by sevoflurane, andthen immediately immersed in heptane, cooled with dry ice to −80° C.,ABR-SOP-0130. The frozen carcasses were embedded in a gel of aqueouscarboxymethyl cellulose (CMC), frozen in ethanol, cooled with dry ice(−80° C.) and sectioned sagittaly for whole body autoradiography,according to the standard method, ABR-SOP-0131. From each animal 20 μmsections were cut at different levels with a cryomicrotome (Leica CM3600) at a temperature of about −20° C. The obtained sections werecaught on tape (Minnesota Mining and Manufacturing Co., No. 810) andnumbered consecutively with radioactive ink. After being freeze-dried at−20° C. for about 24 hours, selected sections were covered with a thinlayer of mylar foil, and put on imaging plates (Fuji, Japan). Exposuretook place in light tight cassettes in a lead shielding box at −20° C.,to protect the image plates from environmental radiation. After exposurethe imaging plates were scanned at a pixel size of 50 μm and analyzed byradioluminography using a bioimaging analysis system (Bas 2500, Fuji,Japan), and described in ABR-SOP-0214. A water-soluble standard testsolution of ³⁵S radioactivity was mixed with whole blood and used forproduction of a calibration scale, ABR-SOP-0251. However, the differentblood standards were dissolved in 500 uL Soluene-35. 4.5 mL Ultima Goldwas then added to the dissolved samples. As ³⁵S and ¹⁴C have verysimilar energy spectra, a standard ¹⁴C-programme (Packard 2200CA) wasused when the radioactivity for the different blood samples was settled.

Pharmacokinetic Calculations:

The ³⁵S radioactivity measured in whole blood and tissues was expressedas nCi/g tissue and recalculated to nmol equiv/g tissue for thepharmacokinetic evaluation. The pharmacokinetic parameters C_(max),t_(1/2) and AUC were determined for the whole blood and tissues bynon-compartmental analysis using WinNonlin Professional (PharsightCorporation, Mountain View, Calif., USA). After intravenousadministration, the concentration was extrapolated back to zero andexpressed as (C₀). The elimination rate constant λ was estimated bylinear regression analysis of the terminal slope of the logarithmicplasma concentration-time curve. The elimination half-life, tin, wascalculated using the equation, t_(1/2)=ln 2/λ. The last threetime-points above LOQ were used in the elimination half-lifecalculations, if not stated otherwise.

Example 38 Assessment of Let-7 Inhibition In Vivo by an 8-MerLNA-antimiR, as Determined Through Ras Protein Quantification in MouseLung and Kidney

In order to investigate the possibility to antagonize the abundantlyexpressed let-7 family in vivo, mice were intravenously (i.v.) injectedwith an 8-mer LNA-antimiR antagonist or with saline. To measuretreatment effect, proteins were isolated from lungs and kidneys. Becausethe Ras family of proteins (N-Ras, K-Ras, and H-Ras), in particularN-Ras and K-Ras, has previously been shown to be regulated (repressed)by the let-7 family by Johnson et al. (Cell, 2005), the aim was toanalyze whether these let-7 targets could be de-repressed in vivo.

Results:

As seen in FIG. 37, the 8-mer LNA-antimiR potently de-repressed Rasprotein levels in the kidneys of treated mice, normalized against salinecontrols. The up-regulation in this organ was more than 3-fold, showinga clear in vivo effect. In the lungs, however, only a minimal (1.2-fold)Ras de-repression was observed (FIG. 1B), suggesting that insufficientamounts of LNA-antimiR has entered this organ in order to inhibit itsmassive amounts of let-7, as previously described by Johnson et al.(Cancer Research, 2007).

Conclusion:

The 8-mer LNA-antimiR shows a clear effect in regulating target let-7miRNA in vivo, as evaluated based on Ras protein levels in treated vs.control mice. Whereas the effect seems to be smaller in lungs, Raslevels in the kidney show a substantial up-regulation uponantimiRs-treatment.

Materials and Methods:

Animals and Dosing:

C57BL/6 female mice were treated with 10 mg/kg LNA-antimiR or saline forthree consecutive days (0, 1, and 2) and sacrificed on day 4. Tissuesamples from lungs and kidneys were snapfrozen and stored at −80° C.until further processing.

Western Blot Analysis:

Lung and kidney proteins from saline and LNA-antimiR-treated mice wereseparated on NuPAGE Bis Tris 4-12% (Invitrogen), using 100 μg persample. The proteins were transferred to a nitrocellulose membrane usingiBlot (Invitrogen) according to the manufacturer's instructions.Blocking, antibody dilution and detection was performed according to themanufacturer's specifications. For Ras detection, a primary rabbit-antiRas antibody (SC-3339, Santa Cruz Biotechnology) and a secondaryHRP-conjugated swine-anti-rabbit antibody (P0399, Dako) was used, andfor tubulin detection, a primary tubulin alpha (MS-581-P1, Neomarkers)and a secondary HRP-conjugated goat-anti-mouse antibody (P0447, Dako)was used.

Example 40 In Vivo Efficacy Assessment of the 8-Mer LNA-antimiR(Compound 3205, SEQ ID NO: 2) in Targeting miR-21, as Determined byPdcd4 Protein Up-Regulation in Mouse Kidney

We have shown that an 8-mer LNA-antimiR that is fully LNA-modifiedantagonizes miR-21 and has the ability to regulate the protein levels ofthe miR-21 target Pdcd4 in vitro. We therefore injected the LNA-antimiRinto mice to determine the effects of the LNA-antimiR in vivo. The micereceived 25 mg/kg of Compound 3205 (SEQ ID NO: 2) by i.v. injectionevery other day for 14 days (a total of 5 doses). The mice weresacrificed on day 14, the kidney was removed, and protein was isolated.In order to determine target regulation, Western blot analysis wasperformed.

Results:

As shown in FIG. 38, treating mice with Compound 3205 (SEQ ID NO: 2)showed significantly increased Pdcd4 protein levels as compared to thesaline control. While the normalized Pdcd4 versus Gapdh ratio wasconsistent in both saline samples, the protein up-regulation in the twoLNA-antimiR-treated with Compound 3205 (SEQ ID NO: 2) mice were measuredto 3.3- and 6.3-fold, respectively, demonstrating an in vivopharmacological effect of the Compound 3205 (SEQ ID NO: 2) 8-merLNA-antimiR.

Conclusion:

The fully LNA-modified 8-mer LNA-antimiR Compound 3205 (SEQ ID NO: 2)antagonizes miR-21 in vivo, as demonstrated through its ability tode-repress (up-regulate) mouse kidney levels of Pdcd4, a validatedmiR-21 target.

Materials and Methods:

Animals and Dosing:

C57BL/6 female mice with average of 20 g body weight at first dosingwere used in all experiments and received regular chow diet (Altromin no1324, Brogaarden, Gentofte, Denmark). Substances were formulated inphysiological saline (0.9% NaCl). The animals were dozed withLNA-antimiR or saline (0.9% NaCl), receiving an injection of 25 mg/kgevery other day for 14 days, a total of 5 doses. Animals were sacrificedon day 14.

Western Blot Analysis:

80 μg kidney tissue from saline or LNA-treated mice was separated onNuPAGE Bis Tris 4-12% (Invitrogen). The proteins were transferred to anitrocellulose membrane using iBlot (Invitrogen) according to themanufacturer's instructions. The membrane was incubated with Pdcd4antibody (Bethyl Laboratories), followed by HRP-conjugatedswine-anti-rabbit antibody (Dako). As equal loading control, GAPDH(Abcam) was used, followed by HRP-conjugated swine-anti-mouse antibody.The membranes were visualized by chemiluminescence (ECL, Amersham).

SEQ 9-mer microRNA MicroRNASequence ID NO Compound 8-mer Compound 7-merCompound ebv-miR-BART1-3p UAGCACCGCUAUCCACUAUGUC 40 AGCGGTGCT GCGGTGCTCGGTGCT ebv-miR-BART1-5p UCUUAGUGGAAGUGACGUGCUGUG 41 TCCACTAAG CCACTAAGCACTAAG ebv-miR-BART10 UACAUAACCAUGGAGUUGGCUGU 42 TGGTTATGT GGTTATGTGTTATGT ebv-miR-BART10* GCCACCUCUUUGGUUCUGUACA 43 AAGAGGTGG AGAGGTGGGAGGTGG ebv-miR-BART11- ACGCACACCAGGCUGACUGCC 44 TGGTGTGCG GGTGTGCGGTGTGCG 3p ebv-miR-BART11- UCAGACAGUUUGGUGCGCUAGUUG 45 AACTGTCTGACTGTCTG CTGTCTG 5p ebv-miR-BART12 UCCUGUGGUGUUUGGUGUGGUU 46 CACCACAGGACCACAGG CCACAGG ebv-miR-BART13 UGUAACUUGCCAGGGACGGCUGA 47 GCAAGTTACCAAGTTAC AAGTTAC ebv-miR-BART13* AACCGGCUCGUGGCUCGUACAG 48 CGAGCCGGTGAGCCGGT AGCCGGT ebv-miR-BART14 UAAAUGCUGCAGUAGUAGGGAU 49 GCAGCATTTCAGCATTT AGCATTT ebv-miR-BART14* UACCCUACGCUGCCGAUUUACA 50 GCGTAGGGTCGTAGGGT GTAGGGT ebv-miR-BART15 GUCAGUGGUUUUGUUUCCUUGA 51 AACCACTGAACCACTGA CCACTGA ebv-miR-BART16 UUAGAUAGAGUGGGUGUGUGCUCU 52 CTCTATCTATCTATCTA CTATCTA ebv-miR-BART17- UGUAUGCCUGGUGUCCCCUUAGU 53 CAGGCATACAGGCATAC GGCATAC 3p ebv-miR-BART17- UAAGAGGACGCAGGCAUACAAG 54 CGTCCTCTTGTCCTCTT TCCTCTT 5p ebv-miR-BART18- UAUCGGAAGUUUGGGCUUCGUC 55 ACTTCCGATCTTCCGAT TTCCGAT 3p ebv-miR-BART18- UCAAGUUCGCACUUCCUAUACA 56 GCGAACTTGCGAACTTG GAACTTG 5p ebv-miR-BART19- UUUUGUUUGCUUGGGAAUGCU 57 GCAAACAAACAAACAAA AAACAAA 3p ebv-miR-BART19- ACAUUCCCCGCAAACAUGACAUG 58 CGGGGAATGGGGGAATG GGGAATG 5p ebv-miR-BART2-3p AAGGAGCGAUUUGGAGAAAAUAAA 59ATCGCTCCT TCGCTCCT CGCTCCT ebv-miR-BART2-5p UAUUUUCUGCAUUCGCCCUUGC 60GCAGAAAAT CAGAAAAT AGAAAAT ebv-miR-BART20- CAUGAAGGCACAGCCUGUUACC 61TGCCTTCAT GCCTTCAT CCTTGAT 3p ebv-miR-BART20- UAGCAGGCAUGUCUUCAUUCC 62ATGCCTGCT TGCCTGCT GCCTGCT 5p ebv-miR-BART3 CGCACCACUAGUCACCAGGUGU 63TAGTGGTGC AGTGGTGC GTGGTGC ebv-miR-BART3* ACCUAGUGUUAGUGUUGUGCU 64AACACTAGG ACACTAGG CACTAGG ebv-miR-BART4 GACCUGAUGCUGCUGGUGUGCU 65GCATCAGGT CATCAGGT ATCAGGT ebv-miR-BART5 CAAGGUGAAUAUAGCUGCCCAUCG 66ATTCACCTT TTCACCTT TCACCTT ebv-miR-BART6-3p CGGGGAUCGGACUAGCCUUAGA 67CCGATCCCC CGATCCCC GATCCCC ebv-miR-BART6-5p UAAGGUUGGUCCAAUCCAUAGG 68ACCAACCTT CCAACCTT CAACCTT ebv-miR-BART7 CAUCAUAGUCCAGUGUCCAGGG 69GACTATGAT ACTATGAT CTATGAT ebv-miR-BART7* CCUGGACCUUGACUAUGAAACA 70AAGGTCCAG AGGTCCAG GGTCCAG ebv-miR-BART8 UACGGUUUCCUAGAUUGUACAG 71GGAAACCGT GAAACCGT AAACCGT ebv-miR-BART8* GUCACAAUCUAUGGGGUCGUAGA 72AGATTGTGA GATTGTGA ATTGTGA ebv-miR-BART9 UAACACUUCAUGGGUCCCGUAGU 73TGAAGTGTT GAAGTGTT AAGTGTT ebv-miR-BART9* UACUGGACCCUGAAUUGGAAAC 74GGGTCCAGT GGTCCAGT GTCCAGT ebv-miR-BHRF1-1 UAACCUGAUCAGCCCCGGAGUU 75GATCAGGTT ATCAGGTT TCAGGTT ebv-miR-BHRF1-2 UAUCUUUUGCGGCAGAAAUUGA 76GCAAAAGAT CAAAAGAT AAAAGAT ebv-miR-BHRF1-2* AAAUUCUGUUGCAGCAGAUAGC 77AACAGAATT ACAGAATT CAGAATT ebv-miR-BHRF1-3 UAACGGGAAGUGUGUAAGCACA 78CTTCCCGTT TTCCCGTT TCCCGTT hcmv-miR-UL112 AAGUGACGGUGAGAUCCAGGCU 79ACCGTCACT CCGTCACT CGTCACT hcmv-miR-UL148D UCGUCCUCCCCUUCUUCACCG 80GGGAGGACG GGAGGACG GAGGACG hcmv-miR-UL22A UAACUAGCCUUCCCGUGAGA 81AGGCTAGTT GGCTAGTT GCTAGTT hcmv-miR-UL22A* UCACCAGAAUGCUAGUUUGUAG 82ATTCTGGTG TTCTGGTG TCTGGTG hcmv-miR-UL36 UCGUUGAAGACACCUGGAAAGA 83TCTTCAACG CTTCAACG TTCAACG hcmv-miR-UL36* UUUCCAGGUGUUUUCAACGUGC 84CACCTGGAA ACCTGGAA CCTGGAA hcmv-miR-UL70-3p GGGGAUGGGCUGGCGCGCGG 85GCCCATCCC CCCATCCC CCATCCC hcmv-miR-UL70-5p UGCGUCUCGGCCUCGUCCAGA 86CCGAGACGC CGAGACGC GAGACGC hcmv-miR-US25-1 AACCGCUCAGUGGCUCGGACC 87CTGAGCGGT TGAGCGGT GAGCGGT hcmv-miR-US25-1* UCCGAACGCUAGGUCGGUUCUC 88AGCGTTCGG GCGTTCGG CGTTCGG hcmv-miR-US25-2- AUCCACUUGGAGAGCUCCCGCGG 89CCAAGTGGA CAAGTGGA AAGTGGA 3p hcmv-miR-US25-2- AGCGGUCUGUUCAGGUGGAUGA 90ACAGACCGC CAGACCGC AGACCGC 5p hcmv-miR-US33-3p UCACGGUCCGAGCACAUCCA 91CGGACCGTG GGACCGTG GACCGTG hcmv-miR-US33-5p GAUUGUGCCCGGACCGUGGGCG 92GGGCACAAT GGCACAAT GCACAAT hcmv-miR-US4 CGACAUGGACGUGCAGGGGGAU 93GTCCATGTC TCCATGTC CCATGTC hcmv-miR-US5-1 UGACAAGCCUGACGAGAGCGU 94AGGCTTGTC GGCTTGTC GCTTGTC hcmv-miR-US5-2 UUAUGAUAGGUGUGACGAUGUC 95CCTATCATA CTATCATA TATCATA hsa-let-7a UGAGGUAGUAGGUUGUAUAGUU 96TACTACCTC ACTACCTC CTACCTC (SEQ ID NO: 31) (SEQ ID NO: 25) (SEQ ID NO:30) hsa-let-7a* CUAUACAAUCUACUGUCUUUC 97 GATTGTATA ATTGTATA TTGTATAhsa-let-7b UGAGGUAGUAGGUUGUGUGGUU 98 TACTACCTC ACTACCTC CTACCTC (SEQ ID(SEQ ID NO: 1003) (SEQ ID NO: 1004) NO: 1002) hsa-let-7b*CUAUACAACCUACUGCCUUCCC 99 GGTTGTATA GTTGTATA TTGTATA hsa-let-7cUGAGGUAGUAGGUUGUAUGGUU 100 TACTACCTC ACTACCTC CTACCTC (SEQ ID (SEQ IDNO: 1007) (SEQ ID NO: 1008) NO: 1006) hsa-let-7c* UAGAGUUACACCCUGGGAGUUA101 TGTAACTCT GTAACTCT TAACTCT hsa-let-7d AGAGGUAGUAGGUUGCAUAGUU 102TACTACCTC ACTACCTC CTACCTC (SEQ ID (SEQ ID NO: 1011) (SEQ ID NO: 1012)NO: 1010) hsa-let-7d* CUAUACGACCUGCUGCCUUUCU 103 GGTCGTATA GTCGTATATCGTATA hsa-let-7e UGAGGUAGGAGGUUGUAUAGUU 104 TCCTACCTC CCTACCTC CTACCTC(SEQ ID (SEQ ID NO: 1015) (SEQ ID NO: 1016) NO: 1014) hsa-let-7e*CUAUACGGCCUCCUAGCUUUCC 105 GGCCGTATA GCCGTATA CCGTATA hsa-let-7fUGAGGUAGUAGAUUGUAUAGUU 106 TACTACCTC ACTACCTC CTACCTC (SEQ ID (SEQ IDNO: 1019) (SEQ ID NO: 1020) NO: 1018) hsa-let-7f-1*CUAUACAAUCUAUUGCCUUCCC 107 GATTGTATA ATTGTATA TTGTATA hsa-let-7f-2*CUAUACAGUCUACUGUCUUUCC 108 GACTGTATA ACTGTATA CTGTATA hsa-let-7gUGAGGUAGUAGUUUGUACAGUU 109 TACTACCTC ACTACCTC CTACCTC (SEQ ID (SEQ IDNO: 1023) (SEQ ID NO: 1024) NO: 1022) hsa-let-7g* CUGUACAGGCCACUGCCUUGC110 GCCTGTACA CCTGTACA CTGTACA hsa-let-7i UGAGGUAGUAGUUUGUGCUGUU 111TACTACCTC ACTACCTC CTACCTC (SEQ ID (SEQ ID NO: 1027) (SEQ ID NO: 1028)NO: 1026) hsa-let-7i* CUGCGCAAGCUACUGCCUUGCU 112 GCTTGCGCA CTTGCGCATTGCGCA hsa-miR-1 UGGAAUGUAAAGAAGUAUGUAU 113 TTACATTCC TACATTCC ACATTCChsa-miR-100 AACCCGUAGAUCCGAACUUGUG 114 TCTACGGGT CTACGGGT TACGGGThsa-miR-100* CAAGCUUGUAUCUAUAGGUAUG 115 TACAAGCTT ACAAGCTT CAAGCTThsa-miR-101 UACAGUACUGUGAUAACUGAA 116 CAGTACTGT AGTACTGT GTACTGThsa-miR-101* CAGUUAUCACAGUGCUGAUGCU 117 GTGATAACT TGATAACT GATAACThsa-miR-103 AGCAGCAUUGUACAGGGCUAUGA 118 CAATGCTGC AATGCTGC ATGCTGChsa-miR-103-as UCAUAGCCCUGUACAAUGCUGCU 119 AGGGCTATG GGGCTATG GGCTATGhsa-miR-105 UCAAAUGCUCAGACUCCUGUGGU 120 GAGCATTTG AGCATTTG GCATTTGhsa-miR-105* ACGGAUGUUUGAGCAUGUGCUA 121 AAACATCCG AACATCCG ACATCCGhsa-miR-106a AAAAGUGCUUACAGUGCAGGUAG 122 AAGCACTTT AGCACTTT GCACTTThsa-miR-106a* CUGCAAUGUAAGCACUUCUUAC 123 TACATTGCA ACATTGCA CATTGCAhsa-miR-106b UAAAGUGCUGACAGUGCAGAU 124 CAGCACTTT AGCACTTT GCACTTT (SEQID NO: 989) (SEQ ID NO: 19) (SEQ ID NO: 990) hsa-miR-106b*CCGCACUGUGGGUACUUGCUGC 125 CACAGTGCG ACAGTGCG CAGTGCG hsa-miR-107AGCAGCAUUGUACAGGGCUAUCA 126 CAATGCTGC AATGCTGC ATGCTGC hsa-miR-10aUACCCUGUAGAUCCGAAUUUGUG 127 CTACAGGGT TACAGGGT ACAGGGT hsa-miR-10a*CAAAUUCGUAUCUAGGGGAAUA 128 TACGAATTT ACGAATTT CGAATTT hsa-miR-10bUACCCUGUAGAACCGAAUUUGUG 129 CTACAGGGT TACAGGGT ACAGGGT hsa-miR-10b*ACAGAUUCGAUUCUAGGGGAAU 130 TCGAATCTG CGAATCTG GAATCTG hsa-miR-1178UUGCUCACUGUUCUUCCCUAG 131 CAGTGAGCA AGTGAGCA GTGAGCA hsa-miR-1179AAGCAUUCUUUCAUUGGUUGG 132 AAGAATGCT AGAATGCT GAATGCT hsa-miR-1180UUUCCGGCUCGCGUGGGUGUGU 133 GAGCCGGAA AGCCGGAA GCCGGAA hsa-miR-1181CCGUCGCCGCCACCCGAGCCG 134 GCGGCGACG CGGCGACG GGCGACG hsa-miR-1182GAGGGUCUUGGGAGGGAUGUGAC 135 CAAGACCCT AAGACCCT AGACCCT hsa-miR-1183CACUGUAGGUGAUGGUGAGAGUGGGCA 136 ACCTACAGT CCTACAGT CTACAGT hsa-miR-1184CCUGCAGCGACUUGAUGGCUUCC 137 TCGCTGCAG CGCTGCAG GCTGCAG hsa-miR-1185AGAGGAUACCCUUUGUAUGUU 138 GGTATCCTC GTATCCTC TATCCTC hsa-miR-1197UAGGACACAUGGUCUACUUCU 139 ATGTGTCCT TGTGTCCT GTGTCCT hsa-miR-1200CUCCUGAGCCAUUCUGAGCCUC 140 GGCTCAGGA GCTCAGGA CTCAGGA hsa-miR-1201AGCCUGAUUAAACACAUGCUCUGA 141 TAATCAGGC AATCAGGC ATCAGGC hsa-miR-1202GUGCCAGCUGCAGUGGGGGAG 142 CAGCTGGCA AGCTGGCA GCTGGCA hsa-miR-1203CCCGGAGCCAGGAUGCAGCUC 143 TGGCTCCGG GGCTCCGG GCTCCGG hsa-miR-1204UCGUGGCCUGGUCUCCAUUAU 144 CAGGCCACG AGGCCACG GGCCACG hsa-miR-1205UCUGCAGGGUUUGCUUUGAG 145 ACCCTGCAG CCCTGCAG CCTGCAG hsa-miR-1206UGUUCAUGUAGAUGUUUAAGC 146 TACATGAAC ACATGAAC CATGAAC hsa-miR-1207-3pUCAGCUGGCCCUCAUUUC 147 GGCCAGCTG GCCAGCTG CCAGCTG hsa-miR-1207-5pUGGCAGGGAGGCUGGGAGGGG 148 CTCCCTGCC TCCCTGCC CCCTGCC hsa-miR-1208UCACUGUUCAGACAGGCGGA 149 TGAACAGTG GAACAGTG AACAGTG hsa-miR-122UGGAGUGUGACAAUGGUGUUUG 150 TCACACTCC CACACTCC ACACTCC (SEQ ID NO: 982)(SEQ ID NO: 983) (SEQ ID NO: 984) hsa-miR-122* AACGCCAUUAUCACACUAAAUA151 TAATGGCGT AATGGCGT ATGGCGT hsa-miR-1224-3p CCCCACCUCCUCUCUCCUCAG 152GGAGGTGGG GAGGTGGG AGGTGGG hsa-miR-1224-5p GUGAGGACUCGGGAGGUGG 153GAGTCCTCA AGTCCTCA GTCCTCA hsa-miR-1225-3p UGAGCCCCUGUGCCGCCCCCAG 154CAGGGGCTC AGGGGCTC GGGGCTC hsa-miR-1225-5p GUGGGUACGGCCCAGUGGGGGG 155CCGTACCCA CGTACCCA GTACCCA hsa-miR-1226 UCACCAGCCCUGUGUUCCCUAG 156GGGCTGGTG GGCTGGTG GCTGGTG hsa-miR-1226* GUGAGGGCAUGCAGGCCUGGAUGGGG 157ATGCCCTCA TGCCCTCA GCCCTCA hsa-miR-1227 CGUGCCACCCUUUUCCCCAG 158GGGTGGCAC GGTGGCAC GTGGCAC hsa-miR-1228 UCACACCUGCCUCGCCCCCC 159GCAGGTGTG CAGGTGTG AGGTGTG hsa-miR-1228* GUGGGCGGGGGCAGGUGUGUG 160CCCCGCCCA CCCGCCCA CCGCCCA hsa-miR-1229 CUCUCACCACUGCCCUCCCACAG 161GTGGTGAGA TGGTGAGA GGTGAGA hsa-miR-1231 GUGUCUGGGCGGACAGCUGC 162GCCCAGACA CCCAGACA CCAGACA hsa-miR-1233 UGAGCCCUGUCCUCCCGCAG 163ACAGGGCTC CAGGGCTC AGGGCTC hsa-miR-1234 UCGGCCUGACCACCCACCCCAC 164GTCAGGCCG TCAGGCCG CAGGCCG hsa-miR-1236 CCUCUUCCCCUUGUCUCUCCAG 165GGGGAAGAG GGGAAGAG GGAAGAG hsa-miR-1237 UCCUUCUGCUCCGUCCCCCAG 166AGCAGAAGG GCAGAAGG CAGAAGG hsa-miR-1238 CUUCCUCGUCUGUCUGCCCC 167GACGAGGAA ACGAGGAA CGAGGAA hsa-miR-124 UAAGGCACGCGGUGAAUGCC 168GCGTGCCTT CGTGCCTT GTGCCTT hsa-miR-124* CGUGUUCACAGCGGACCUUGAU 169TGTGAACAC GTGAACAC TGAACAC hsa-miR-1243 AACUGGAUCAAUUAUAGGAGUG 170TGATCCAGT GATCCAGT ATCCAGT hsa-miR-1244 AAGUAGUUGGUUUGUAUGAGAUGGUU 171CCAACTACT CAACTACT AACTACT hsa-miR-1245 AAGUGAUCUAAAGGCCUACAU 172TAGATCACT AGATCACT GATCACT hsa-miR-1246 AAUGGAUUUUUGGAGCAGG 173AAAATCCAT AAATCCAT AATCCAT hsa-miR-1247 ACCCGUCCCGUUCGUCCCCGGA 174CGGGACGGG GGGACGGG GGACGGG hsa-miR-1248 ACCUUCUUGUAUAAGCACUGUGCUAAA 175ACAAGAAGG CAAGAAGG AAGAAGG hsa-miR-1249 ACGCCCUUCCCCCCCUUCUUCA 176GGAAGGGCG GAAGGGCG AAGGGCG hsa-miR-1250 ACGGUGCUGGAUGUGGCCUUU 177CCAGCACCG CAGCACCG AGCACCG hsa-miR-1251 ACUCUAGCUGCCAAAGGCGCU 178CAGCTAGAG AGCTAGAG GCTAGAG hsa-miR-1252 AGAAGGAAAUUGAAUUCAUUUA 179ATTTCCTTC TTTCCTTC TTCCTTC hsa-miR-1253 AGAGAAGAAGAUCAGCCUGCA 180CTTCTTCTC TTCTTCTC TCTTCTC hsa-miR-1254 AGCCUGGAAGCUGGAGCCUGCAGU 181CTTCCAGGC TTCCAGGC TCCAGGC hsa-miR-1255a AGGAUGAGCAAAGAAAGUAGAUU 182TGCTCATCC GCTCATCC CTCATCC hsa-miR-1255b CGGAUGAGCAAAGAAAGUGGUU 183TGCTCATCC GCTCATCC CTCATCC hsa-miR-1256 AGGCAUUGACUUCUCACUAGCU 184GTCAATGCC TCAATGCC CAATGCC hsa-miR-1257 AGUGAAUGAUGGGUUCUGACC 185ATCATTCAC TCATTCAC CATTCAC hsa-miR-1258 AGUUAGGAUUAGGUCGUGGAA 186AATCCTAAC ATCCTAAC TCCTAAC hsa-miR-1259 AUAUAUGAUGACUUAGCUUUU 187CATCATATA ATCATATA TCATATA hsa-miR-125a-3p ACAGGUGAGGUUCUUGGGAGCC 188CCTCACCTG CTCACCTG TCACCTG hsa-miR-125a-5p UCCCUGAGACCCUUUAACCUGUGA 189GTCTCAGGG TCTCAGGG CTCAGGG hsa-miR-125b UCCCUGAGACCCUAACUUGUGA 190GTCTCAGGG TCTCAGGG CTCAGGG hsa-miR-125b-1* ACGGGUUAGGCUCUUGGGAGCU 191CCTAACCCG CTAACCCG TAACCCG hsa-miR-125b-2* UCACAAGUCAGGCUCUUGGGAC 192TGACTTGTG GACTTGTG ACTTGTG hsa-miR-126 UCGUACCGUGAGUAAUAAUGCG 193CACGGTACG ACGGTACG CGGTACG hsa-miR-126* CAUUAUUACUUUUGGUACGCG 194AGTAATAAT GTAATAAT TAATAAT hsa-miR-1260 AUCCCACCUCUGCCACCA 195 GAGGTGGGAAGGTGGGA GGTGGGA hsa-miR-1261 AUGGAUAAGGCUUUGGCUU 196 CCTTATCCA CTTATCCATTATCCA hsa-miR-1262 AUGGGUGAAUUUGUAGAAGGAU 197 ATTCACCCA TTCACCCATCACCCA hsa-miR-1263 AUGGUACCCUGGCAUACUGAGU 198 AGGGTACCA GGGTACCAGGTACCA hsa-miR-1264 CAAGUCUUAUUUGAGCACCUGUU 199 ATAAGACTT TAAGACTTAAGACTT hsa-miR-1265 CAGGAUGUGGUCAAGUGUUGUU 200 CCACATCCT CACATCCTACATCCT hsa-miR-1266 CCUCAGGGCUGUAGAACAGGGCU 201 AGCCCTGAG GCCCTGAGCCCTGAG hsa-miR-1267 CCUGUUGAAGUGUAAUCCCCA 202 CTTCAACAG TTCAACAGTCAACAG hsa-miR-1268 CGGGCGUGGUGGUGGGGG 203 ACCACGCCC CCACGCCC CACGCCChsa-miR-1269 CUGGACUGAGCCGUGCUACUGG 204 CTCAGTCCA TCAGTCCA CAGTCCAhsa-miR-127-3p UCGGAUCCGUCUGAGCUUGGCU 205 ACGGATCCG CGGATCCG GGATCCGhsa-miR-127-5p CUGAAGCUCAGAGGGCUCUGAU 206 TGAGCTTCA GAGCTTCA AGCTTCAhsa-miR-1270 CUGGAGAUAUGGAAGAGCUGUGU 207 ATATCTCCA TATCTCCA ATCTCCAhsa-miR-1271 CUUGGCACCUAGCAAGCACUCA 208 AGGTGCCAA GGTGCCAA GTGCCAAhsa-miR-1272 GAUGAUGAUGGCAGCAAAUUCUGAAA 209 CATCATCAT ATCATCAT TCATCAThsa-miR-1273 GGGCGACAAAGCAAGACUCUUUCUU 210 TTTGTCGCC TTGTCGCC TGTCGCChsa-miR-1274a GUCCCUGUUCAGGCGCCA 211 GAACAGGGA AACAGGGA ACAGGGAhsa-miR-1274b UCCCUGUUCGGGCGCCA 212 CGAACAGGG GAACAGGG AACAGGGhsa-miR-1275 GUGGGGGAGAGGCUGUC 213 TCTCCCCCA CTCCCCCA TCCCCCAhsa-miR-1276 UAAAGAGCCCUGUGGAGACA 214 GGGCTCTTT GGCTCTTT GCTCTTThsa-miR-1277 UACGUAGAUAUAUAUGUAUUUU 215 TATCTACGT ATCTACGT TCTACGThsa-miR-1278 UAGUACUGUGCAUAUCAUCUAU 216 CACAGTACT ACAGTACT CAGTACThsa-miR-1279 UCAUAUUGCUUCUUUCU 217 AGCAATATG GCAATATG CAATATGhsa-miR-128 UCACAGUGAACCGGUCUCUUU 218 TTCACTGTG TCACTGTG CACTGTGhsa-miR-1280 UCCCACCGCUGCCACCC 219 AGCGGTGGG GCGGTGGG CGGTGGGhsa-miR-1281 UCGCCUCCUCCUCUCCC 220 GAGGAGGCG AGGAGGCG GGAGGCGhsa-miR-1282 UCGUUUGCCUUUUUCUGCUU 221 AGGCAAACG GGCAAACG GCAAACGhsa-miR-1283 UCUACAAAGGAAAGCGCUUUCU 222 CCTTTGTAG CTTTGTAG TTTGTAGhsa-miR-1284 UCUAUACAGACCCUGGCUUUUC 223 TCTGTATAG CTGTATAG TGTATAGhsa-miR-1285 UCUGGGCAACAAAGUGAGACCU 224 GTTGCCCAG TTGCCCAG TGCCCAGhsa-miR-1286 UGCAGGACCAAGAUGAGCCCU 225 TGGTCCTGC GGTCCTGC GTCCTGChsa-miR-1287 UGCUGGAUCAGUGGUUCGAGUC 226 TGATCCAGC GATCCAGC ATCCAGChsa-miR-1288 UGGACUGCCCUGAUCUGGAGA 227 GGGCAGTCC GGCAGTCC GCAGTCChsa-miR-1289 UGGAGUCCAGGAAUCUGCAUUUU 228 CTGGACTCC TGGACTCC GGACTCChsa-miR-129* AAGCCCUUACCCCAAAAAGUAU 229 GTAAGGGCT TAAGGGCT AAGGGCThsa-miR-129-3p AAGCCCUUACCCCAAAAAGCAU 230 GTAAGGGCT TAAGGGCT AAGGGCThsa-miR-129-5p CUUUUUGCGGUCUGGGCUUGC 231 CCGCAAAAA CGCAAAAA GCAAAAAhsa-miR-1290 UGGAUUUUUGGAUCAGGGA 232 CAAAAATCC AAAAATCC AAAATCChsa-miR-1291 UGGCCCUGACUGAAGACCAGCAGU 233 GTCAGGGCC TCAGGGCC CAGGGCChsa-miR-1292 UGGGAACGGGUUCCGGCAGACGCUG 234 CCCGTTCCC CCGTTCCC CGTTCCChsa-miR-1293 UGGGUGGUCUGGAGAUUUGUGC 235 AGACCACCC GACCACCC ACCACCChsa-miR-1294 UGUGAGGUUGGCAUUGUUGUCU 236 CAACCTCAC AACCTCAC ACCTCAChsa-miR-1295 UUAGGCCGCAGAUCUGGGUGA 237 TGCGGCCTA GCGGCCTA CGGCCTAhsa-miR-1296 UUAGGGCCCUGGCUCCAUCUCC 238 AGGGCCCTA GGGCCCTA GGCCCTAhsa-miR-1297 UUCAAGUAAUUCAGGUG 239 ATTACTTGA TTACTTGA TACTTGAhsa-miR-1298 UUCAUUCGGCUGUCCAGAUGUA 240 GCCGAATGA CCGAATGA CGAATGAhsa-miR-1299 UUCUGGAAUUCUGUGUGAGGGA 241 AATTCCAGA ATTCCAGA TTCCAGAhsa-miR-1300 UUGAGAAGGAGGCUGCUG 242 TCCTTCTCA CCTTCTCA CTTCTCAhsa-miR-1301 UUGCAGCUGCCUGGGAGUGACUUC 243 GCAGCTGCA CAGCTGCA AGCTGCAhsa-miR-1302 UUGGGACAUACUUAUGCUAAA 244 TATGTCCCA ATGTCCCA TGTCCCAhsa-miR-1303 UUUAGAGACGGGGUCUUGCUCU 245 CGTCTCTAA GTCTCTAA TCTCTAAhsa-miR-1304 UUUGAGGCUACAGUGAGAUGUG 246 TAGCCTCAA AGCCTCAA GCCTCAAhsa-miR-1305 UUUUCAACUCUAAUGGGAGAGA 247 GAGTTGAAA AGTTGAAA GTTGAAAhsa-miR-1306 ACGUUGGCUCUGGUGGUG 248 GAGCCAACG AGCCAACG GCCAACGhsa-miR-1307 ACUCGGCGUGGCGUCGGUCGUG 249 CACGCCGAG ACGCCGAG CGCCGAGhsa-miR-1308 GCAUGGGUGGUUCAGUGG 250 CCACCCATG CACCCATG ACCCATGhsa-miR-130a CAGUGCAAUGUUAAAAGGGCAU 251 CATTGCACT ATTGCACT TTGCACThsa-miR-130a* UUCACAUUGUGCUACUGUCUGC 252 ACAATGTGA CAATGTGA AATGTGAhsa-miR-130b CAGUGCAAUGAUGAAAGGGCAU 253 CATTGCACT ATTGCACT TTGCACThsa-miR-130b* ACUCUUUCCCUGUUGCACUAC 254 GGGAAAGAG GGAAAGAG GAAAGAGhsa-miR-132 UAACAGUCUACAGCCAUGGUCG 255 TAGACTGTT AGACTGTT GACTGTThsa-miR-132* ACCGUGGCUUUCGAUUGUUACU 256 AAGCCACGG AGCCACGG GCCACGGhsa-miR-1321 CAGGGAGGUGAAUGUGAU 257 CACCTCCCT ACCTCCCT CCTCCCThsa-miR-1322 GAUGAUGCUGCUGAUGCUG 258 CAGCATCAT AGCATCAT GCATCAThsa-miR-1323 UCAAAACUGAGGGGCAUUUUCU 259 TCAGTTTTG CAGTTTTG AGTTTTGhsa-miR-1324 CCAGACAGAAUUCUAUGCACUUUC 260 TTCTGTCTG TCTGTCTG CTGTCTGhsa-miR-133a UUUGGUCCCCUUCAACCAGCUG 261 GGGGACCAA GGGACCAA GGACCAAhsa-miR-133b UUUGGUCCCCUUCAACCAGCUA 262 GGGGACCAA GGGACCAA GGACCAAhsa-miR-134 UGUGACUGGUUGACCAGAGGGG 263 ACCAGTCAC CCAGTCAC CAGTCAChsa-miR-135a UAUGGCUUUUUAUUCCUAUGUGA 264 AAAAGCCAT AAAGCCAT AAGCCAThsa-miR-135a* UAUAGGGAUUGGAGCCGUGGCG 265 AATCCCTAT ATCCCTAT TCCCTAThsa-miR-135b UAUGGCUUUUCAUUCCUAUGUGA 266 AAAAGCCAT AAAGCCAT AAGCCAThsa-miR-135b* AUGUAGGGCUAAAAGCCAUGGG 267 AGCCCTACA GCCCTACA CCCTACAhsa-miR-136 ACUCCAUUUGUUUUGAUGAUGGA 268 CAAATGGAG AAATGGAG AATGGAGhsa-miR-136* CAUCAUCGUCUCAAAUGAGUCU 269 GACGATGAT ACGATGAT CGATGAThsa-miR-137 UUAUUGCUUAAGAAUACGCGUAG 270 TAAGCAATA AAGCAATA AGCAATAhsa-miR-138 AGCUGGUGUUGUGAAUCAGGCCG 271 AACACCAGC ACACCAGC CACCAGChsa-miR-138-1* GCUACUUCACAACACCAGGGCC 272 GTGAAGTAG TGAAGTAG GAAGTAGhsa-miR-138-2* GCUAUUUCACGACACCAGGGUU 273 GTGAAATAG TGAAATAG GAAATAGhsa-miR-139-3p GGAGACGCGGCCCUGUUGGAGU 274 CCGCGTCTC CGCGTCTC GCGTCTChsa-miR-139-5p UCUACAGUGCACGUGUCUCCAG 275 GCACTGTAG CACTGTAG ACTGTAGhsa-miR-140-3p UACCACAGGGUAGAACCACGG 276 CCCTGTGGT CCTGTGGT CTGTGGThsa-miR-140-5p CAGUGGUUUUACCCUAUGGUAG 277 AAAACCACT AAACCACT AACCACThsa-miR-141 UAACACUGUCUGGUAAAGAUGG 278 GACAGTGTT ACAGTGTT CAGTGTThsa-miR-141* CAUCUUCCAGUACAGUGUUGGA 279 CTGGAAGAT TGGAAGAT GGAAGAThsa-miR-142-3p UGUAGUGUUUCCUACUUUAUGGA 280 AAACACTAC AACACTAC ACACTAChsa-miR-142-5p CAUAAAGUAGAAAGCACUACU 281 CTACTTTAT TACTTTAT ACTTTAThsa-miR-143 UGAGAUGAAGCACUGUAGCUC 282 CTTCATCTC TTCATCTC TCATCTChsa-miR-143* GGUGCAGUGCUGCAUCUCUGGU 283 GCACTGCAC CACTGCAC ACTGCAChsa-miR-144 UACAGUAUAGAUGAUGUACU 284 CTATACTGT TATACTGT ATACTGThsa-miR-144* GGAUAUCAUCAUAUACUGUAAG 285 GATGATATC ATGATATC TGATATChsa-miR-145 GUCCAGUUUUCCCAGGAAUCCCU 286 AAAACTGGA AAACTGGA AACTGGAhsa-miR-145* GGAUUCCUGGAAAUACUGUUCU 287 CCAGGAATC CAGGAATC AGGAATChsa-miR-1468 CUCCGUUUGCCUGUUUCGCUG 288 GCAAACGGA CAAACGGA AAACGGAhsa-miR-1469 CUCGGCGCGGGGCGCGGGCUCC 289 CCGCGCCGA CGCGCCGA GCGCCGAhsa-miR-146a UGAGAACUGAAUUCCAUGGGUU 290 TCAGTTCTC CAGTTCTC AGTTCTChsa-miR-146a* CCUCUGAAAUUCAGUUCUUCAG 291 ATTTCAGAG TTTCAGAG TTCAGAGhsa-miR-146b-3p UGCCCUGUGGACUCAGUUCUGG 292 CCACAGGGC CACAGGGC ACAGGGChsa-miR-146b-5p UGAGAACUGAAUUCCAUAGGCU 293 TCAGTTCTC CAGTTCTC AGTTCTChsa-miR-147 GUGUGUGGAAAUGCUUCUGC 294 TTCCACACA TCCACACA CCACACAhsa-miR-1470 GCCCUCCGCCCGUGCACCCCG 295 GGCGGAGGG GCGGAGGG CGGAGGGhsa-miR-1471 GCCCGCGUGUGGAGCCAGGUGU 296 ACACGCGGG CACGCGGG ACGCGGGhsa-miR-147b GUGUGCGGAAAUGCUUCUGCUA 297 TTCCGCACA TCCGCACA CCGCACAhsa-miR-148a UCAGUGCACUACAGAACUUUGU 298 AGTGCACTG GTGCACTG TGCACTGhsa-miR-148a* AAAGUUCUGAGACACUCCGACU 299 TCAGAACTT CAGAACTT AGAACTThsa-miR-148b UCAGUGCAUCACAGAACUUUGU 300 GATGCACTG ATGCACTG TGCACTGhsa-miR-148b* AAGUUCUGUUAUACACUCAGGC 301 AACAGAACT ACAGAACT CAGAACThsa-miR-149 UCUGGCUCCGUGUCUUCACUCCC 302 CGGAGCCAG GGAGCCAG GAGCCAGhsa-miR-149* AGGGAGGGACGGGGGCUGUGC 303 GTCCCTCCC TCCCTCCC CCCTCCChsa-miR-150 UCUCCCAACCCUUGUACCAGUG 304 GGTTGGGAG GTTGGGAG TTGGGAGhsa-miR-150* CUGGUACAGGCCUGGGGGACAG 305 CCTGTACCA CTGTACCA TGTACCAhsa-miR-151-3p CUAGACUGAAGCUCCUUGAGG 306 TTCAGTCTA TCAGTCTA CAGTCTAhsa-miR-151-5p UCGAGGAGCUCACAGUCUAGU 307 AGCTCCTCG GCTCCTCG CTCCTCGhsa-miR-152 UCAGUGCAUGACAGAACUUGG 308 CATGCACTG ATGCACTG TGCACTGhsa-miR-153 UUGCAUAGUCACAAAAGUGAUC 309 GACTATGCA ACTATGCA CTATGCAhsa-miR-1537 AAAACCGUCUAGUUACAGUUGU 310 AGACGGTTT GACGGTTT ACGGTTThsa-miR-1538 CGGCCCGGGCUGCUGCUGUUCCU 311 GCCCGGGCC CCCGGGCC CCGGGCChsa-miR-1539 UCCUGCGCGUCCCAGAUGCCC 312 ACGCGCAGG CGCGCAGG GCGCAGGhsa-miR-154 UAGGUUAUCCGUGUUGCCUUCG 313 GGATAACCT GATAACCT ATAACCThsa-miR-154* AAUCAUACACGGUUGACCUAUU 314 GTGTATGAT TGTATGAT GTATGAThsa-miR-155 UUAAUGCUAAUCGUGAUAGGGGU 315 TTAGCATTA TAGCATTA AGCATTA (SEQID NO: 986) (SEQ ID NO: 4) (SEQ ID NO: 987) hsa-miR-155*CUCCUACAUAUUAGCAUUAACA 316 TATGTAGGA ATGTAGGA TGTAGGA hsa-miR-15aUAGCAGCACAUAAUGGUUUGUG 317 TGTGCTGCT GTGCTGCT TGCTGCT hsa-miR-15a*CAGGCCAUAUUGUGCUGCCUCA 318 ATATGGCCT TATGGCCT ATGGCCT hsa-miR-15bUAGCAGCACAUCAUGGUUUACA 319 TGTGCTGCT GTGCTGCT TGCTGCT hsa-miR-15b*CGAAUCAUUAUUUGCUGCUCUA 320 TAATGATTC AATGATTC ATGATTC hsa-miR-16UAGCAGCACGUAAAUAUUGGCG 321 CGTGCTGCT GTGCTGCT TGCTGCT hsa-miR-16-1*CCAGUAUUAACUGUGCUGCUGA 322 TTAATACTG TAATACTG AATACTG hsa-miR-16-2*CCAAUAUUACUGUGCUGCUUUA 323 GTAATATTG TAATATTG AATATTG hsa-miR-17CAAAGUGCUUACAGUGCAGGUAG 324 AAGCACTTT AGCACTTT GCACTTT hsa-miR-17*ACUGCAGUGAAGGCACUUGUAG 325 TCACTGCAG CACTGCAG ACTGCAG hsa-miR-181aAACAUUCAACGCUGUCGGUGAGU 326 GTTGAATGT TTGAATGT TGAATGT hsa-miR-181a*ACCAUCGACCGUUGAUUGUACC 327 GGTCGATGG GTCGATGG TCGATGG hsa-miR-181a-2*ACCACUGACCGUUGACUGUACC 328 GGTCAGTGG GTCAGTGG TCAGTGG hsa-miR-181bAACAUUCAUUGCUGUCGGUGGGU 329 AATGAATGT ATGAATGT TGAATGT hsa-miR-181cAACAUUCAACCUGUCGGUGAGU 330 GTTGAATGT TTGAATGT TGAATGT hsa-miR-181c*AACCAUCGACCGUUGAGUGGAC 331 GTCGATGGT TCGATGGT CGATGGT hsa-miR-181dAACAUUCAUUGUUGUCGGUGGGU 332 AATGAATGT ATGAATGT TGAATGT hsa-miR-182UUUGGCAAUGGUAGAACUCACACU 333 CATTGCCAA ATTGCCAA TTGCCAA hsa-miR-182*UGGUUCUAGACUUGCCAACUA 334 TCTAGAACC CTAGAACC TAGAACC hsa-miR-1825UCCAGUGCCCUCCUCUCC 335 GGGCACTGG GGCACTGG GCACTGG hsa-miR-1826AUUGAUCAUCGACACUUCGAACGCAAU 336 GATGATCAA ATGATCAA TGATCAA hsa-miR-1827UGAGGCAGUAGAUUGAAU 337 TACTGCCTC ACTGCCTC CTGCCTC hsa-miR-183UAUGGCACUGGUAGAAUUCACU 338 CAGTGCCAT AGTGCCAT GTGCCAT hsa-miR-183*GUGAAUUACCGAAGGGCCAUAA 339 GGTAATTCA GTAATTCA TAATTCA hsa-miR-184UGGACGGAGAACUGAUAAGGGU 340 TCTCCGTCC CTCCGTCC TCCGTCC hsa-miR-185UGGAGAGAAAGGCAGUUCCUGA 341 TTTCTCTCC TTCTCTCC TCTCTCC hsa-miR-185*AGGGGCUGGCUUUCCUCUGGUC 342 GCCAGCCCC CCAGCCCC CAGCCCC hsa-miR-186CAAAGAAUUCUCCUUUUGGGCU 343 GAATTCTTT AATTCTTT ATTCTTT hsa-miR-186*GCCCAAAGGUGAAUUUUUUGGG 344 ACCTTTGGG CCTTTGGG CTTTGGG hsa-miR-187UCGUGUCUUGUGUUGCAGCCGG 345 CAAGACACG AAGACACG AGACACG hsa-miR-187*GGCUACAACACAGGACCCGGGC 346 TGTTGTAGC GTTGTAGC TTGTAGC hsa-miR-188-3pCUCCCACAUGCAGGGUUUGCA 347 CATGTGGGA ATGTGGGA TGTGGGA hsa-miR-188-5pCAUCCCUUGCAUGGUGGAGGG 348 GCAAGGGAT CAAGGGAT AAGGGAT hsa-miR-18aUAAGGUGCAUCUAGUGCAGAUAG 349 ATGCACCTT TGCACCTT GCACCTT hsa-miR-18a*ACUGCCCUAAGUGCUCCUUCUGG 350 TTAGGGCAG TAGGGCAG AGGGCAG hsa-miR-18bUAAGGUGCAUCUAGUGCAGUUAG 351 ATGCACCTT TGCACCTT GCACCTT hsa-miR-18b*UGCCCUAAAUGCCCCUUCUGGC 352 ATTTAGGGC TTTAGGGC TTAGGGC hsa-miR-190UGAUAUGUUUGAUAUAUUAGGU 353 AAACATATC AACATATC ACATATC hsa-miR-1908CGGCGGGGACGGCGAUUGGUC 354 GTCCCCGCC TCCCCGCC CCCCGCC hsa-miR-1909CGCAGGGGCCGGGUGCUCACCG 355 GGCCCCTGC GCCCCTGC CCCCTGC hsa-miR-1909*UGAGUGCCGGUGCCUGCCCUG 356 CCGGCACTC CGGCACTC GGCACTC hsa-miR-190bUGAUAUGUUUGAUAUUGGGUU 357 AAACATATC AACATATC ACATATC hsa-miR-191CAACGGAAUCCCAAAAGCAGCUG 358 GATTCCGTT ATTCCGTT TTCCGTT hsa-miR-191*GCUGCGCUUGGAUUUCGUCCCC 359 CAAGCGCAG AAGCGCAG AGCGCAG hsa-miR-1910CCAGUCCUGUGCCUGCCGCCU 360 ACAGGACTG CAGGACTG AGGACTG hsa-miR-1911UGAGUACCGCCAUGUCUGUUGGG 361 GCGGTACTC CGGTACTC GGTACTC hsa-miR-1911*CACCAGGCAUUGUGGUCUCC 362 ATGCCTGGT TGCCTGGT GCCTGGT hsa-miR-1912UACCCAGAGCAUGCAGUGUGAA 363 GCTCTGGGT CTCTGGGT TCTGGGT hsa-miR-1913UCUGCCCCCUCCGCUGCUGCCA 364 AGGGGGCAG GGGGGCAG GGGGCAG hsa-miR-1914CCCUGUGCCCGGCCCACUUCUG 365 GGGCACAGG GGCACAGG GCACAGG hsa-miR-1914*GGAGGGGUCCCGCACUGGGAGG 366 GGACCCCTC GACCCCTC ACCCCTC hsa-miR-1915CCCCAGGGCGACGCGGCGGG 367 CGCCCTGGG GCCCTGGG CCCTGGG hsa-miR-1915*ACCUUGCCUUGCUGCCCGGGCC 368 AAGGCAAGG AGGCAAGG GGCAAGG hsa-miR-192CUGACCUAUGAAUUGACAGCC 369 CATAGGTCA ATAGGTCA TAGGTCA hsa-miR-192*CUGCCAAUUCCAUAGGUCACAG 370 GAATTGGCA AATTGGCA ATTGGCA hsa-miR-193a-3pAACUGGCCUACAAAGUCCCAGU 371 TAGGCCAGT AGGCCAGT GGCCAGT hsa-miR-193a-5pUGGGUCUUUGCGGGCGAGAUGA 372 CAAAGACCC AAAGACCC AAGACCC hsa-miR-193bAACUGGCCCUCAAAGUCCCGCU 373 AGGGCCAGT GGGCCAGT GGCCAGT hsa-miR-193b*CGGGGUUUUGAGGGCGAGAUGA 374 CAAAACCCC AAAACCCC AAACCCC hsa-miR-194UGUAACAGCAACUCCAUGUGGA 375 TGCTGTTAC GCTGTTAC CTGTTAC hsa-miR-194*CCAGUGGGGCUGCUGUUAUCUG 376 GCCCCACTG CCCCACTG CCCACTG hsa-miR-195UAGCAGCACAGAAAUAUUGGC 377 TGTGCTGCT GTGCTGCT TGCTGCT hsa-miR-195*CCAAUAUUGGCUGUGCUGCUCC 378 CCAATATTG CAATATTG AATATTG hsa-miR-196aUAGGUAGUUUCAUGUUGUUGGG 379 AAACTACCT AACTACCT ACTACCT hsa-miR-196a*CGGCAACAAGAAACUGCCUGAG 380 CTTGTTGCC TTGTTGCC TGTTGCC hsa-miR-196bUAGGUAGUUUCCUGUUGUUGGG 381 AAACTACCT AACTACCT ACTACCT hsa-miR-197UUCACCACCUUCUCCACCCAGC 382 AGGTGGTGA GGTGGTGA GTGGTGA hsa-miR-198GGUCCAGAGGGGAGAUAGGUUC 383 CCTCTGGAC CTCTGGAC TCTGGAC hsa-miR-199a-5pCCCAGUGUUCAGACUACCUGUUC 384 GAACACTGG AACACTGG ACACTGG hsa-miR-199b-3pACAGUAGUCUGCACAUUGGUUA 385 AGACTACTG GACTACTG ACTACTG hsa-miR-199b-5pCCCAGUGUUUAGACUAUCUGUUC 386 AAACACTGG AACACTGG ACACTGG hsa-miR-19aUGUGCAAAUCUAUGCAAAACUGA 387 GATTTGCAC ATTTGCAC TTTGCAC hsa-miR-19a*AGUUUUGCAUAGUUGCACUACA 388 ATGCAAAAC TGCAAAAC GCAAAAC hsa-miR-19bUGUGCAAAUCCAUGCAAAACUGA 389 GATTTGCAC ATTTGCAC TTTGCAC (SEQ ID NO: 992)(SEQ ID NO: 20) (SEQ ID NO: 993) hsa-miR-19b-1* AGUUUUGCAGGUUUGCAUCCAGC390 CTGCAAAAC TGCAAAAC GCAAAAC hsa-miR-19b-2* AGUUUUGCAGGUUUGCAUUUCA 391CTGCAAAAC TGCAAAAC GCAAAAC hsa-miR-200a UAACACUGUCUGGUAACGAUGU 392GACAGTGTT ACAGTGTT CAGTGTT hsa-miR-200a* CAUCUUACCGGACAGUGCUGGA 393CGGTAAGAT GGTAAGAT GTAAGAT hsa-miR-200b UAAUACUGCCUGGUAAUGAUGA 394GGCAGTATT GCAGTATT CAGTATT hsa-miR-200b* CAUCUUACUGGGCAGCAUUGGA 395CAGTAAGAT AGTAAGAT GTAAGAT hsa-miR-200c UAAUACUGCCGGGUAAUGAUGGA 396GGCAGTATT GCAGTATT CAGTATT hsa-miR-200c* CGUCUUACCCAGCAGUGUUUGG 397GGGTAAGAC GGTAAGAC GTAAGAC hsa-miR-202 AGAGGUAUAGGGCAUGGGAA 398CTATACCTC TATACCTC ATACCTC hsa-miR-202* UUCCUAUGCAUAUACUUCUUUG 399TGCATAGGA GCATAGGA CATAGGA hsa-miR-203 GUGAAAUGUUUAGGACCACUAG 400AACATTTCA ACATTTCA CATTTCA hsa-miR-204 UUCCCUUUGUCAUCCUAUGCCU 401ACAAAGGGA CAAAGGGA AAAGGGA hsa-miR-205 UCCUUCAUUCCACCGGAGUCUG 402GAATGAAGG AATGAAGG ATGAAGG hsa-miR-206 UGGAAUGUAAGGAAGUGUGUGG 403TTACATTCC TACATTCC ACATTCC hsa-miR-208a AUAAGACGAGCAAAAAGCUUGU 404CTCGTCTTA TCGTCTTA CGTCTTA hsa-miR-208b AUAAGACGAACAAAAGGUUUGU 405TTCGTCTTA TCGTCTTA CGTCTTA hsa-miR-20a UAAAGUGCUUAUAGUGCAGGUAG 406AAGCACTTT AGCACTTT GCACTTT hsa-miR-20a* ACUGCAUUAUGAGCACUUAAAG 407ATAATGCAG TAATGCAG AATGCAG hsa-miR-20b CAAAGUGCUCAUAGUGCAGGUAG 408GAGCACTTT AGCACTTT GCACTTT hsa-miR-20b* ACUGUAGUAUGGGCACUUCCAG 409ATACTACAG TACTACAG ACTACAG hsa-miR-21 UAGCUUAUCAGACUGAUGUUGA 410TGATAAGCT GATAAGCT ATAAGCT (SEQ ID NO: 9) (SEQ ID NO: 2) (SEQ ID NO: 8)hsa-miR-21* CAACACCAGUCGAUGGGCUGU 411 ACTGGTGTT CTGGTGTT TGGTGTThsa-miR-210 CUGUGCGUGUGACAGCGGCUGA 412 ACACGCACA CACGCACA ACGCACAhsa-miR-211 UUCCCUUUGUCAUCCUUCGCCU 413 ACAAAGGGA CAAAGGGA AAAGGGAhsa-miR-212 UAACAGUCUCCAGUCACGGCC 414 GAGACTGTT AGACTGTT GACTGTThsa-miR-214 ACAGCAGGCACAGACAGGCAGU 415 TGCCTGCTG GCCTGCTG CCTGCTGhsa-miR-214* UGCCUGUCUACACUUGCUGUGC 416 TAGACAGGC AGACAGGC GACAGGChsa-miR-215 AUGACCUAUGAAUUGACAGAC 417 CATAGGTCA ATAGGTCA TAGGTCAhsa-miR-216a UAAUCUCAGCUGGCAACUGUGA 418 GCTGAGATT CTGAGATT TGAGATThsa-miR-216b AAAUCUCUGCAGGCAAAUGUGA 419 GCAGAGATT CAGAGATT AGAGATThsa-miR-217 UACUGCAUCAGGAACUGAUUGGA 420 TGATGCAGT GATGCAGT ATGCAGThsa-miR-218 UUGUGCUUGAUCUAACCAUGU 421 TCAAGCACA CAAGCACA AAGCACAhsa-miR-218-1* AUGGUUCCGUCAAGCACCAUGG 422 ACGGAACCA CGGAACCA GGAACCAhsa-miR-218-2* CAUGGUUCUGUCAAGCACCGCG 423 CAGAACCAT AGAACCAT GAACCAThsa-miR-219-1-3p AGAGUUGAGUCUGGACGUCCCG 424 ACTCAACTC CTCAACTC TCAACTChsa-miR-219-2-3p AGAAUUGUGGCUGGACAUCUGU 425 CCACAATTC CACAATTC ACAATTChsa-miR-219-5p UGAUUGUCCAAACGCAAUUCU 426 TGGACAATC GGACAATC GACAATChsa-miR-22 AAGCUGCCAGUUGAAGAACUGU 427 CTGGCAGCT TGGCAGCT GGCAGCThsa-miR-22* AGUUCUUCAGUGGCAAGCUUUA 428 CTGAAGAAC TGAAGAAC GAAGAAChsa-miR-220a CCACACCGUAUCUGACACUUU 429 TACGGTGTG ACGGTGTG CGGTGTGhsa-miR-220b CCACCACCGUGUCUGACACUU 430 ACGGTGGTG CGGTGGTG GGTGGTGhsa-miR-220c ACACAGGGCUGUUGUGAAGACU 431 AGCCCTGTG GCCCTGTG CCCTGTGhsa-miR-221 AGCUACAUUGUCUGCUGGGUUUC 432 CAATGTAGC AATGTAGC ATGTAGC (SEQID NO: 995) (SEQ ID NO: 996) (SEQ ID NO: 23) hsa-miR-221*ACCUGGCAUACAAUGUAGAUUU 433 TATGCCAGG ATGCCAGG TGCCAGG hsa-miR-222AGCUACAUCUGGCUACUGGGU 434 AGATGTAGC GATGTAGC ATGTAGC (SEQ ID NO: 998)(SEQ ID NO: 999) (SEQ ID NO: 23) hsa-miR-222* CUCAGUAGCCAGUGUAGAUCCU 435GGCTACTGA GCTACTGA CTACTGA hsa-miR-223 UGUCAGUUUGUCAAAUACCCCA 436CAAACTGAC AAACTGAC AACTGAC hsa-miR-223* CGUGUAUUUGACAAGCUGAGUU 437CAAATACAC AAATACAC AATACAC hsa-miR-224 CAAGUCACUAGUGGUUCCGUU 438TAGTGACTT AGTGACTT GTGACTT hsa-miR-23a AUCACAUUGCCAGGGAUUUCC 439GCAATGTGA CAATGTGA AATGTGA hsa-miR-23a* GGGGUUCCUGGGGAUGGGAUUU 440CAGGAACCC AGGAACCC GGAACCC hsa-miR-23b AUCACAUUGCCAGGGAUUACC 441GCAATGTGA CAATGTGA AATGTGA hsa-miR-23b* UGGGUUCCUGGCAUGCUGAUUU 442CAGGAACCC AGGAACCC GGAACCC hsa-miR-24 UGGCUCAGUUCAGCAGGAACAG 443AACTGAGCC ACTGAGCC CTGAGCC hsa-miR-24-1* UGCCUACUGAGCUGAUAUCAGU 444TCAGTAGGC CAGTAGGC AGTAGGC hsa-miR-24-2* UGCCUACUGAGCUGAAACACAG 445TCAGTAGGC CAGTAGGC AGTAGGC hsa-miR-25 CAUUGCACUUGUCUCGGUCUGA 446AAGTGCAAT AGTGCAAT GTGCAAT hsa-miR-25* AGGCGGAGACUUGGGCAAUUG 447GTCTCCGCC TCTCCGCC CTCCGCC hsa-miR-26a UUCAAGUAAUCCAGGAUAGGCU 448ATTACTTGA TTACTTGA TACTTGA hsa-miR-26a-1* CCUAUUCUUGGUUACUUGCACG 449CAAGAATAG AAGAATAG AGAATAG hsa-miR-26a-2* CCUAUUCUUGAUUACUUGUUUC 450CAAGAATAG AAGAATAG AGAATAG hsa-miR-26b UUCAAGUAAUUCAGGAUAGGU 451ATTACTTGA TTACTTGA TACTTGA hsa-miR-26b* CCUGUUCUCCAUUACUUGGCUC 452GGAGAACAG GAGAACAG AGAACAG hsa-miR-27a UUCACAGUGGCUAAGUUCCGC 453CCACTGTGA CACTGTGA ACTGTGA hsa-miR-27a* AGGGCUUAGCUGCUUGUGAGCA 454GCTAAGCCC CTAAGCCC TAAGCCC hsa-miR-27b UUCACAGUGGCUAAGUUCUGC 455CCACTGTGA CACTGTGA ACTGTGA hsa-miR-27b* AGAGCUUAGCUGAUUGGUGAAC 456GCTAAGCTC CTAAGCTC TAAGCTC hsa-miR-28-3p CACUAGAUUGUGAGCUCCUGGA 457CAATCTAGT AATCTAGT ATCTAGT hsa-miR-28-5p AAGGAGCUCACAGUCUAUUGAG 458TGAGCTCCT GAGCTCCT AGCTCCT hsa-miR-296-3p GAGGGUUGGGUGGAGGCUCUCC 459CCCAACCCT CCAACCCT CAACCCT hsa-miR-296-5p AGGGCCCCCCCUCAAUCCUGU 460GGGGGGCCC GGGGGCCC GGGGCCC hsa-miR-297 AUGUAUGUGUGCAUGUGCAUG 461ACACATACA CACATACA ACATACA hsa-miR-298 AGCAGAAGCAGGGAGGUUCUCCCA 462TGCTTCTGC GCTTCTGC CTTCTGC hsa-miR-299-3p UAUGUGGGAUGGUAAACCGCUU 463ATCCCACAT TCCCACAT CCCACAT hsa-miR-299-5p UGGUUUACCGUCCCACAUACAU 464CGGTAAACC GGTAAACC GTAAACC hsa-miR-29a UAGCACCAUCUGAAAUCGGUUA 465GATGGTGCT ATGGTGCT TGGTGCT hsa-miR-29a* ACUGAUUUCUUUUGGUGUUCAG 466AGAAATCAG GAAATCAG AAATCAG hsa-miR-29b UAGCACCAUUUGAAAUCAGUGUU 467AATGGTGCT ATGGTGCT TGGTGCT hsa-miR-29b-1* GCUGGUUUCAUAUGGUGGUUUAGA 468TGAAACCAG GAAACCAG AAACCAG hsa-miR-29b-2* CUGGUUUCACAUGGUGGCUUAG 469GTGAAACCA TGAAACCA GAAACCA hsa-miR-29c UAGCACCAUUUGAAAUCGGUUA 470AATGGTGCT ATGGTGCT TGGTGCT hsa-miR-29c* UGACCGAUUUCUCCUGGUGUUC 471AAATCGGTC AATCGGTC ATCGGTC hsa-miR-300 UAUACAAGGGCAGACUCUCUCU 472CCCTTGTAT CCTTGTAT CTTGTAT hsa-miR-301a CAGUGCAAUAGUAUUGUCAAAGC 473TATTGCACT ATTGCACT TTGCACT hsa-miR-301b CAGUGCAAUGAUAUUGUCAAAGC 474CATTGCACT ATTGCACT TTGCACT hsa-miR-302a UAAGUGCUUCCAUGUUUUGGUGA 475GAAGCACTT AAGCACTT AGCACTT hsa-miR-302a* ACUUAAACGUGGAUGUACUUGCU 476ACGTTTAAG CGTTTAAG GTTTAAG hsa-miR-302b UAAGUGCUUCCAUGUUUUAGUAG 477GAAGCACTT AAGCACTT AGCACTT hsa-miR-302b* ACUUUAACAUGGAAGUGCUUUC 478ATGTTAAAG TGTTAAAG GTTAAAG hsa-miR-302c UAAGUGCUUCCAUGUUUCAGUGG 479GAAGCACTT AAGCACTT AGCACTT hsa-miR-302c* UUUAACAUGGGGGUACCUGCUG 480CCATGTTAA CATGTTAA ATGTTAA hsa-miR-302d UAAGUGCUUCCAUGUUUGAGUGU 481GAAGCACTT AAGCACTT AGCACTT hsa-miR-302d* ACUUUAACAUGGAGGCACUUGC 482ATGTTAAAG TGTTAAAG GTTAAAG hsa-miR-302e UAAGUGCUUCCAUGCUU 483 GAAGCACTTAAGCACTT AGCACTT hsa-miR-302f UAAUUGCUUCCAUGUUU 484 GAAGCAATT AAGCAATTAGCAATT hsa-miR-30a UGUAAACAUCCUCGACUGGAAG 485 GATGTTTAC ATGTTTACTGTTTAC hsa-miR-30a* CUUUCAGUCGGAUGUUUGCAGC 486 CGACTGAAA GACTGAAAACTGAAA hsa-miR-30b UGUAAACAUCCUACACUCAGCU 487 GATGTTTAC ATGTTTACTGTTTAC hsa-miR-30b* CUGGGAGGUGGAUGUUUACUUC 488 CACCTCCCA ACCTCCCACCTCCCA hsa-miR-30c UGUAAACAUCCUACACUCUCAGC 489 GATGTTTAC ATGTTTACTGTTTAC hsa-miR-30c-1* CUGGGAGAGGGUUGUUUACUCC 490 CCTCTCCCA CTCTCCCATCTCCCA hsa-miR-30c-2* CUGGGAGAAGGCUGUUUACUCU 491 CTTCTCCCA TTCTCCCATCTCCCA hsa-miR-30d UGUAAACAUCCCCGACUGGAAG 492 GATGTTTAC ATGTTTACTGTTTAC hsa-miR-30d* CUUUCAGUCAGAUGUUUGCUGC 493 TGACTGAAA GACTGAAAACTGAAA hsa-miR-30e UGUAAACAUCCUUGACUGGAAG 494 GATGTTTAC ATGTTTACTGTTTAC hsa-miR-30e* CUUUCAGUCGGAUGUUUACAGC 495 CGACTGAAA GACTGAAAACTGAAA hsa-miR-31 AGGCAAGAUGCUGGCAUAGCU 496 CATCTTGCC ATCTTGCC TCTTGCChsa-miR-31* UGCUAUGCCAACAUAUUGCCAU 497 TGGCATAGC GGCATAGC GCATAGChsa-miR-32 UAUUGCACAUUACUAAGUUGCA 498 ATGTGCAAT TGTGCAAT GTGCAAThsa-miR-32* CAAUUUAGUGUGUGUGAUAUUU 499 CACTAAATT ACTAAATT CTAAATThsa-miR-320a AAAAGCUGGGUUGAGAGGGCGA 500 CCCAGCTTT CCAGCTTT CAGCTTThsa-miR-320b AAAAGCUGGGUUGAGAGGGCAA 501 CCCAGCTTT CCAGCTTT CAGCTTThsa-miR-320c AAAAGCUGGGUUGAGAGGGU 502 CCCAGCTTT CCAGCTTT CAGCTTThsa-miR-320d AAAAGCUGGGUUGAGAGGA 503 CCCAGCTTT CCAGCTTT CAGCTTThsa-miR-323-3p CACAUUACACGGUCGACCUCU 504 GTGTAATGT TGTAATGT GTAATGThsa-miR-323-5p AGGUGGUCCGUGGCGCGUUCGC 505 CGGACCACC GGACCACC GACCACChsa-miR-324-3p ACUGCCCCAGGUGCUGCUGG 506 CTGGGGCAG TGGGGCAG GGGGCAGhsa-miR-324-5p CGCAUCCCCUAGGGCAUUGGUGU 507 AGGGGATGC GGGGATGC GGGATGChsa-miR-325 CCUAGUAGGUGUCCAGUAAGUGU 508 ACCTACTAG CCTACTAG CTACTAGhsa-miR-326 CCUCUGGGCCCUUCCUCCAG 509 GGCCCAGAG GCCCAGAG CCCAGAGhsa-miR-328 CUGGCCCUCUCUGCCCUUCCGU 510 AGAGGGCCA GAGGGCCA AGGGCCAhsa-miR-329 AACACACCUGGUUAACCUCUUU 511 CAGGTGTGT AGGTGTGT GGTGTGThsa-miR-330-3p GCAAAGCACACGGCCUGCAGAGA 512 TGTGCTTTG GTGCTTTG TGCTTTGhsa-miR-330-5p UCUCUGGGCCUGUGUCUUAGGC 513 GGCCCAGAG GCCCAGAG CCCAGAGhsa-miR-331-3p GCCCCUGGGCCUAUCCUAGAA 514 GCCCAGGGG CCCAGGGG CCAGGGGhsa-miR-331-5p CUAGGUAUGGUCCCAGGGAUCC 515 CCATACCTA CATACCTA ATACCTAhsa-miR-335 UCAAGAGCAAUAACGAAAAAUGU 516 TTGCTCTTG TGCTCTTG GCTCTTGhsa-miR-335* UUUUUCAUUAUUGCUCCUGACC 517 TAATGAAAA AATGAAAA ATGAAAAhsa-miR-337-3p CUCCUAUAUGAUGCCUUUCUUC 518 CATATAGGA ATATAGGA TATAGGAhsa-miR-337-5p GAACGGCUUCAUACAGGAGUU 519 GAAGCCGTT AAGCCGTT AGCCGTThsa-miR-338-3p UCCAGCAUCAGUGAUUUUGUUG 520 TGATGCTGG GATGCTGG ATGCTGGhsa-miR-338-5p AACAAUAUCCUGGUGCUGAGUG 521 GGATATTGT GATATTGT ATATTGThsa-miR-339-3p UGAGCGCCUCGACGACAGAGCCG 522 GAGGCGCTC AGGCGCTC GGCGCTChsa-miR-339-5p UCCCUGUCCUCCAGGAGCUCACG 523 AGGACAGGG GGACAGGG GACAGGGhsa-miR-33a GUGCAUUGUAGUUGCAUUGCA 524 TACAATGCA ACAATGCA CAATGCAhsa-miR-33a* CAAUGUUUCCACAGUGCAUCAC 525 GGAAACATT GAAACATT AAACATThsa-miR-33b GUGCAUUGCUGUUGCAUUGC 526 AGCAATGCA GCAATGCA CAATGCAhsa-miR-33b* CAGUGCCUCGGCAGUGCAGCCC 527 CGAGGCACT GAGGCACT AGGCACThsa-miR-340 UUAUAAAGCAAUGAGACUGAUU 528 TGCTTTATA GCTTTATA CTTTATAhsa-miR-340* UCCGUCUCAGUUACUUUAUAGC 529 CTGAGACGG TGAGACGG GAGACGGhsa-miR-342-3p UCUCACACAGAAAUCGCACCCGU 530 CTGTGTGAG TGTGTGAG GTGTGAGhsa-miR-342-5p AGGGGUGCUAUCUGUGAUUGA 531 TAGCACCCC AGCACCCC GCACCCChsa-miR-345 GCUGACUCCUAGUCCAGGGCUC 532 AGGAGTCAG GGAGTCAG GAGTCAGhsa-miR-346 UGUCUGCCCGCAUGCCUGCCUCU 533 CGGGCAGAC GGGCAGAC GGCAGAChsa-miR-34a UGGCAGUGUCUUAGCUGGUUGU 534 GACACTGCC ACACTGCC CACTGCChsa-miR-34a* CAAUCAGCAAGUAUACUGCCCU 535 TTGCTGATT TGCTGATT GCTGATThsa-miR-34b CAAUCACUAACUCCACUGCCAU 536 TTAGTGATT TAGTGATT AGTGATThsa-miR-34b* UAGGCAGUGUCAUUAGCUGAUUG 537 ACACTGCCT CACTGCCT ACTGCCThsa-miR-34c-3p AAUCACUAACCACACGGCCAGG 538 GTTAGTGAT TTAGTGAT TAGTGAThsa-miR-34c-5p AGGCAGUGUAGUUAGCUGAUUGC 539 TACACTGCC ACACTGCC CACTGCChsa-miR-361-3p UCCCCCAGGUGUGAUUCUGAUUU 540 ACCTGGGGG CCTGGGGG CTGGGGGhsa-miR-361-5p UUAUCAGAAUCUCCAGGGGUAC 541 ATTCTGATA TTCTGATA TCTGATAhsa-miR-362-3p AACACACCUAUUCAAGGAUUCA 542 TAGGTGTGT AGGTGTGT GGTGTGThsa-miR-362-5p AAUCCUUGGAACCUAGGUGUGAGU 543 TCCAAGGAT CCAAGGAT CAAGGAThsa-miR-363 AAUUGCACGGUAUCCAUCUGUA 544 CCGTGCAAT CGTGCAAT GTGCAAThsa-miR-363* CGGGUGGAUCACGAUGCAAUUU 545 GATCCACCC ATCCACCC TCCACCChsa-miR-365 UAAUGCCCCUAAAAAUCCUUAU 546 AGGGGCATT GGGGCATT GGGCATThsa-miR-367 AAUUGCACUUUAGCAAUGGUGA 547 AAGTGCAAT AGTGCAAT GTGCAAThsa-miR-367* ACUGUUGCUAAUAUGCAACUCU 548 TAGCAACAG AGCAACAG GCAACAGhsa-miR-369-3p AAUAAUACAUGGUUGAUCUUU 549 ATGTATTAT TGTATTAT GTATTAThsa-miR-369-5p AGAUCGACCGUGUUAUAUUCGC 550 CGGTCGATC GGTCGATC GTCGATChsa-miR-370 GCCUGCUGGGGUGGAACCUGGU 551 CCCAGCAGG CCAGCAGG CAGCAGGhsa-miR-371-3p AAGUGCCGCCAUCUUUUGAGUGU 552 GGCGGCACT GCGGCACT CGGCACThsa-miR-371-5p ACUCAAACUGUGGGGGCACU 553 CAGTTTGAG AGTTTGAG GTTTGAGhsa-miR-372 AAAGUGCUGCGACAUUUGAGCGU 554 GCAGCACTT CAGCACTT AGCACTThsa-miR-373 GAAGUGCUUCGAUUUUGGGGUGU 555 GAAGCACTT AAGCACTT AGCACTThsa-miR-373* ACUCAAAAUGGGGGCGCUUUCC 556 CATTTTGAG ATTTTGAG TTTTGAGhsa-miR-374a UUAUAAUACAACCUGAUAAGUG 557 TGTATTATA GTATTATA TATTATAhsa-miR-374a* CUUAUCAGAUUGUAUUGUAAUU 558 ATCTGATAA TCTGATAA CTGATAAhsa-miR-374b AUAUAAUACAACCUGCUAAGUG 559 TGTATTATA GTATTATA TATTATAhsa-miR-374b* CUUAGCAGGUUGUAUUAUCAUU 560 ACCTGCTAA CCTGCTAA CTGCTAAhsa-miR-375 UUUGUUCGUUCGGCUCGCGUGA 561 AACGAACAA ACGAACAA CGAACAAhsa-miR-376a AUCAUAGAGGAAAAUCCACGU 562 CCTCTATGA CTCTATGA TCTATGAhsa-miR-376a* GUAGAUUCUCCUUCUAUGAGUA 563 GAGAATCTA AGAATCTA GAATCTAhsa-miR-376b AUCAUAGAGGAAAAUCCAUGUU 564 CCTCTATGA CTCTATGA TCTATGAhsa-miR-376c AACAUAGAGGAAAUUCCACGU 565 CCTCTATGT CTCTATGT TCTATGThsa-miR-377 AUCACACAAAGGCAACUUUUGU 566 TTTGTGTGA TTGTGTGA TGTGTGAhsa-miR-377* AGAGGUUGCCCUUGGUGAAUUC 567 GGCAACCTC GCAACCTC CAACCTChsa-miR-378 ACUGGACUUGGAGUCAGAAGG 568 CAAGTCCAG AAGTCCAG AGTCCAGhsa-miR-378* CUCCUGACUCCAGGUCCUGUGU 569 GAGTCAGGA AGTCAGGA GTCAGGAhsa-miR-379 UGGUAGACUAUGGAACGUAGG 570 TAGTCTACC AGTCTACC GTCTACChsa-miR-379* UAUGUAACAUGGUCCACUAACU 571 ATGTTACAT TGTTACAT GTTACAThsa-miR-380 UAUGUAAUAUGGUCCACAUCUU 572 ATATTACAT TATTACAT ATTACAThsa-miR-380* UGGUUGACCAUAGAACAUGCGC 573 TGGTCAACC GGTCAACC GTCAACChsa-miR-381 UAUACAAGGGCAAGCUCUCUGU 574 CCCTTGTAT CCTTGTAT CTTGTAThsa-miR-382 GAAGUUGUUCGUGGUGGAUUCG 575 GAACAACTT AACAACTT ACAACTThsa-miR-383 AGAUCAGAAGGUGAUUGUGGCU 576 CTTCTGATC TTCTGATC TCTGATChsa-miR-384 AUUCCUAGAAAUUGUUCAUA 577 TTCTAGGAA TCTAGGAA CTAGGAAhsa-miR-409-3p GAAUGUUGCUCGGUGAACCCCU 578 AGCAACATT GCAACATT CAACATThsa-miR-409-5p AGGUUACCCGAGCAACUUUGCAU 579 CGGGTAACC GGGTAACC GGTAACChsa-miR-410 AAUAUAACACAGAUGGCCUGU 580 GTGTTATAT TGTTATAT GTTATAThsa-miR-411 UAGUAGACCGUAUAGCGUACG 581 CGGTCTACT GGTCTACT GTCTACThsa-miR-411* UAUGUAACACGGUCCACUAACC 582 GTGTTACAT TGTTACAT GTTACAThsa-miR-412 ACUUCACCUGGUCCACUAGCCGU 583 CAGGTGAAG AGGTGAAG GGTGAAGhsa-miR-421 AUCAACAGACAUUAAUUGGGCGC 584 GTCTGTTGA TCTGTTGA CTGTTGAhsa-miR-422a ACUGGACUUAGGGUCAGAAGGC 585 TAAGTCCAG AAGTCCAG AGTCCAGhsa-miR-423-3p AGCUCGGUCUGAGGCCCCUCAGU 586 AGACCGAGC GACCGAGC ACCGAGChsa-miR-423-5p UGAGGGGCAGAGAGCGAGACUUU 587 CTGCCCCTC TGCCCCTC GCCCCTChsa-miR-424 CAGCAGCAAUUCAUGUUUUGAA 588 ATTGCTGCT TTGCTGCT TGCTGCThsa-miR-424* CAAAACGUGAGGCGCUGCUAU 589 TCACGTTTT CACGTTTT ACGTTTThsa-miR-425 AAUGACACGAUCACUCCCGUUGA 590 TCGTGTCAT CGTGTCAT GTGTCAThsa-miR-425* AUCGGGAAUGUCGUGUCCGCCC 591 CATTCCCGA ATTCCCGA TTCCCGAhsa-miR-429 UAAUACUGUCUGGUAAAACCGU 592 GACAGTATT ACAGTATT CAGTATThsa-miR-431 UGUCUUGCAGGCCGUCAUGCA 593 CTGCAAGAC TGCAAGAC GCAAGAChsa-miR-431* CAGGUCGUCUUGCAGGGCUUCU 594 AGACGACCT GACGACCT ACGACCThsa-miR-432 UCUUGGAGUAGGUCAUUGGGUGG 595 TACTCCAAG ACTCCAAG CTCCAAGhsa-miR-432* CUGGAUGGCUCCUCCAUGUCU 596 AGCCATCCA GCCATCCA CCATCCAhsa-miR-433 AUCAUGAUGGGCUCCUCGGUGU 597 CCATCATGA CATCATGA ATCATGAhsa-miR-448 UUGCAUAUGUAGGAUGUCCCAU 598 ACATATGCA CATATGCA ATATGCAhsa-miR-449a UGGCAGUGUAUUGUUAGCUGGU 599 TACACTGCC ACACTGCC CACTGCChsa-miR-449b AGGCAGUGUAUUGUUAGCUGGC 600 TACACTGCC ACACTGCC CACTGCChsa-miR-450a UUUUGCGAUGUGUUCCUAAUAU 601 CATCGCAAA ATCGCAAA TCGCAAAhsa-miR-450b-3p UUGGGAUCAUUUUGCAUCCAUA 602 ATGATCCCA TGATCCCA GATCCCAhsa-miR-450b-5p UUUUGCAAUAUGUUCCUGAAUA 603 TATTGCAAA ATTGCAAA TTGCAAAhsa-miR-451 AAACCGUUACCAUUACUGAGUU 604 GTAACGGTT TAACGGTT AACGGTThsa-miR-452 AACUGUUUGCAGAGGAAACUGA 605 GCAAACAGT CAAACAGT AAACAGThsa-miR-452* CUCAUCUGCAAAGAAGUAAGUG 606 TGCAGATGA GCAGATGA CAGATGAhsa-miR-453 AGGUUGUCCGUGGUGAGUUCGCA 607 CGGACAACC GGACAACC GACAACChsa-miR-454 UAGUGCAAUAUUGCUUAUAGGGU 608 TATTGCACT ATTGCACT TTGCACThsa-miR-454* ACCCUAUCAAUAUUGUCUCUGC 609 TTGATAGGG TGATAGGG GATAGGGhsa-miR-455-3p GCAGUCCAUGGGCAUAUACAC 610 CATGGACTG ATGGACTG TGGACTGhsa-miR-455-5p UAUGUGCCUUUGGACUACAUCG 611 AAGGCACAT AGGCACAT GGCACAThsa-miR-483-3p UCACUCCUCUCCUCCCGUCUU 612 AGAGGAGTG GAGGAGTG AGGAGTGhsa-miR-483-5p AAGACGGGAGGAAAGAAGGGAG 613 CTCCCGTCT TCCCGTCT CCCGTCThsa-miR-484 UCAGGCUCAGUCCCCUCCCGAU 614 CTGAGCCTG TGAGCCTG GAGCCTGhsa-miR-485-3p GUCAUACACGGCUCUCCUCUCU 615 CGTGTATGA GTGTATGA TGTATGAhsa-miR-485-5p AGAGGCUGGCCGUGAUGAAUUC 616 GCCAGCCTC CCAGCCTC CAGCCTChsa-miR-486-3p CGGGGCAGCUCAGUACAGGAU 617 AGCTGCCCC GCTGCCCC CTGCCCChsa-miR-486-5p UCCUGUACUGAGCUGCCCCGAG 618 CAGTACAGG AGTACAGG GTACAGGhsa-miR-487a AAUCAUACAGGGACAUCCAGUU 619 CTGTATGAT TGTATGAT GTATGAThsa-miR-487b AAUCGUACAGGGUCAUCCACUU 620 CTGTACGAT TGTACGAT GTACGAThsa-miR-488 UUGAAAGGCUAUUUCUUGGUC 621 AGCCTTTCA GCCTTTCA CCTTTCAhsa-miR-488* CCCAGAUAAUGGCACUCUCAA 622 ATTATCTGG TTATCTGG TATCTGGhsa-miR-489 GUGACAUCACAUAUACGGCAGC 623 GTGATGTCA TGATGTCA GATGTCAhsa-miR-490-3p CAACCUGGAGGACUCCAUGCUG 624 CTCCAGGTT TCCAGGTT CCAGGTThsa-miR-490-5p CCAUGGAUCUCCAGGUGGGU 625 AGATCCATG GATCCATG ATCCATGhsa-miR-491-3p CUUAUGCAAGAUUCCCUUCUAC 626 CTTGCATAA TTGCATAA TGCATAAhsa-miR-491-5p AGUGGGGAACCCUUCCAUGAGG 627 GTTCCCCAC TTCCCCAC TCCCCAChsa-miR-492 AGGACCUGCGGGACAAGAUUCUU 628 CGCAGGTCC GCAGGTCC CAGGTCChsa-miR-493 UGAAGGUCUACUGUGUGCCAGG 629 TAGACCTTC AGACCTTC GACCTTChsa-miR-493* UUGUACAUGGUAGGCUUUCAUU 630 CCATGTACA CATGTACA ATGTACAhsa-miR-494 UGAAACAUACACGGGAAACCUC 631 GTATGTTTC TATGTTTC ATGTTTChsa-miR-495 AAACAAACAUGGUGCACUUCUU 632 ATGTTTGTT TGTTTGTT GTTTGTThsa-miR-496 UGAGUAUUACAUGGCCAAUCUC 633 GTAATACTC TAATACTC AATACTChsa-miR-497 CAGCAGCACACUGUGGUUUGU 634 TGTGCTGCT GTGCTGCT TGCTGCThsa-miR-497* CAAACCACACUGUGGUGUUAGA 635 GTGTGGTTT TGTGGTTT GTGGTTThsa-miR-498 UUUCAAGCCAGGGGGCGUUUUUC 636 TGGCTTGAA GGCTTGAA GCTTGAAhsa-miR-499-3p AACAUCACAGCAAGUCUGUGCU 637 CTGTGATGT TGTGATGT GTGATGThsa-miR-499-5p UUAAGACUUGCAGUGAUGUUU 638 CAAGTCTTA AAGTCTTA AGTCTTAhsa-miR-500 UAAUCCUUGCUACCUGGGUGAGA 639 GCAAGGATT CAAGGATT AAGGATThsa-miR-500* AUGCACCUGGGCAAGGAUUCUG 640 CCAGGTGCA CAGGTGCA AGGTGCAhsa-miR-501-3p AAUGCACCCGGGCAAGGAUUCU 641 CGGGTGCAT GGGTGCAT GGTGCAThsa-miR-501-5p AAUCCUUUGUCCCUGGGUGAGA 642 ACAAAGGAT CAAAGGAT AAAGGAThsa-miR-502-3p AAUGCACCUGGGCAAGGAUUCA 643 CAGGTGCAT AGGTGCAT GGTGCAThsa-miR-502-5p AUCCUUGCUAUCUGGGUGCUA 644 TAGCAAGGA AGCAAGGA GCAAGGAhsa-miR-503 UAGCAGCGGGAACAGUUCUGCAG 645 CCCGCTGCT CCGCTGCT CGCTGCThsa-miR-504 AGACCCUGGUCUGCACUCUAUC 646 ACCAGGGTC CCAGGGTC CAGGGTChsa-miR-505 CGUCAACACUUGCUGGUUUCCU 647 AGTGTTGAC GTGTTGAC TGTTGAChsa-miR-505* GGGAGCCAGGAAGUAUUGAUGU 648 CCTGGCTCC CTGGCTCC TGGCTCChsa-miR-506 UAAGGCACCCUUCUGAGUAGA 649 GGGTGCCTT GGTGCCTT GTGCCTThsa-miR-507 UUUUGCACCUUUUGGAGUGAA 650 AGGTGCAAA GGTGCAAA GTGCAAAhsa-miR-508-3p UGAUUGUAGCCUUUUGGAGUAGA 651 GCTACAATC CTACAATC TACAATChsa-miR-508-5p UACUCCAGAGGGCGUCACUCAUG 652 CTCTGGAGT TCTGGAGT CTGGAGThsa-miR-509-3-5p UACUGCAGACGUGGCAAUCAUG 653 GTCTGCAGT TCTGCAGT CTGCAGThsa-miR-509-3p UGAUUGGUACGUCUGUGGGUAG 654 GTACCAATC TACCAATC ACCAATChsa-miR-509-5p UACUGCAGACAGUGGCAAUCA 655 GTCTGCAGT TCTGCAGT CTGCAGThsa-miR-510 UACUCAGGAGAGUGGCAAUCAC 656 CTCCTGAGT TCCTGAGT CCTGAGThsa-miR-511 GUGUCUUUUGCUCUGCAGUCA 657 CAAAAGACA AAAAGACA AAAGACAhsa-miR-512-3p AAGUGCUGUCAUAGCUGAGGUC 658 GACAGCACT ACAGCACT CAGCACThsa-miR-512-5p CACUCAGCCUUGAGGGCACUUUC 659 AGGCTGAGT GGCTGAGT GCTGAGThsa-miR-513a-3p UAAAUUUCACCUUUCUGAGAAGG 660 GTGAAATTT TGAAATTT GAAATTThsa-miR-513a-5p UUCACAGGGAGGUGUCAU 661 TCCCTGTGA CCCTGTGA CCTGTGAhsa-miR-513b UUCACAAGGAGGUGUCAUUUAU 662 TCCTTGTGA CCTTGTGA CTTGTGAhsa-miR-513c UUCUCAAGGAGGUGUCGUUUAU 663 TCCTTGAGA CCTTGAGA CTTGAGAhsa-miR-514 AUUGACACUUCUGUGAGUAGA 664 AAGTGTCAA AGTGTCAA GTGTCAAhsa-miR-515-3p GAGUGCCUUCUUUUGGAGCGUU 665 GAAGGCACT AAGGCACT AGGCACThsa-miR-515-5p UUCUCCAAAAGAAAGCACUUUCUG 666 TTTTGGAGA TTTGGAGA TTGGAGAhsa-miR-516a-3p UGCUUCCUUUCAGAGGGU 667 AAAGGAAGC AAGGAAGC AGGAAGChsa-miR-516a-5p UUCUCGAGGAAAGAAGCACUUUC 668 TCCTCGAGA CCTCGAGA CTCGAGAhsa-miR-516b AUCUGGAGGUAAGAAGCACUUU 669 ACCTCCAGA CCTCCAGA CTCCAGAhsa-miR-517* CCUCUAGAUGGAAGCACUGUCU 670 CATCTAGAG ATCTAGAG TCTAGAGhsa-miR-517a AUCGUGCAUCCCUUUAGAGUGU 671 GATGCACGA ATGCACGA TGCACGAhsa-miR-517b UCGUGCAUCCCUUUAGAGUGUU 672 GGATGCACG GATGCACG ATGCACGhsa-miR-517c AUCGUGCAUCCUUUUAGAGUGU 673 GATGCACGA ATGCACGA TGCACGAhsa-miR-518a-3p GAAAGCGCUUCCCUUUGCUGGA 674 AAGCGCTTT AGCGCTTT GCGCTTThsa-miR-518b CAAAGCGCUCCCCUUUAGAGGU 675 GAGCGCTTT AGCGCTTT GCGCTTThsa-miR-518c CAAAGCGCUUCUCUUUAGAGUGU 676 AAGCGCTTT AGCGCTTT GCGCTTThsa-miR-518c* UCUCUGGAGGGAAGCACUUUCUG 677 CCTCCAGAG CTCCAGAG TCCAGAGhsa-miR-518d-3p CAAAGCGCUUCCCUUUGGAGC 678 AAGCGCTTT AGCGCTTT GCGCTTThsa-miR-518d-5p CUCUAGAGGGAAGCACUUUCUG 679 CCCTCTAGA CCTCTAGA CTCTAGAhsa-miR-518e AAAGCGCUUCCCUUCAGAGUG 680 GAAGCGCTT AAGCGCTT AGCGCTThsa-miR-518f GAAAGCGCUUCUCUUUAGAGG 681 AAGCGCTTT AGCGCTTT GCGCTTThsa-miR-518f* CUCUAGAGGGAAGCACUUUCUC 682 CCCTCTAGA CCTCTAGA CTCTAGAhsa-miR-519a AAAGUGCAUCCUUUUAGAGUGU 683 GATGCACTT ATGCACTT TGCACTThsa-miR-519a* CUCUAGAGGGAAGCGCUUUCUG 684 CCCTCTAGA CCTCTAGA CTCTAGAhsa-miR-519b-3p AAAGUGCAUCCUUUUAGAGGUU 685 GATGCACTT ATGCACTT TGCACTThsa-miR-519c-3p AAAGUGCAUCUUUUUAGAGGAU 686 GATGCACTT ATGCACTT TGCACTThsa-miR-519d CAAAGUGCCUCCCUUUAGAGUG 687 AGGCACTTT GGCACTTT GCACTTThsa-miR-519e AAGUGCCUCCUUUUAGAGUGUU 688 GGAGGCACT GAGGCACT AGGCACThsa-miR-519e* UUCUCCAAAAGGGAGCACUUUC 689 TTTTGGAGA TTTGGAGA TTGGAGAhsa-miR-520a-3p AAAGUGCUUCCCUUUGGACUGU 690 GAAGCACTT AAGCACTT AGCACTThsa-miR-520a-5p CUCCAGAGGGAAGUACUUUCU 691 CCCTCTGGA CCTCTGGA CTCTGGAhsa-miR-520b AAAGUGCUUCCUUUUAGAGGG 692 GAAGCACTT AAGCACTT AGCACTThsa-miR-520c-3p AAAGUGCUUCCUUUUAGAGGGU 693 GAAGCACTT AAGCACTT AGCACTThsa-miR-520d-3p AAAGUGCUUCUCUUUGGUGGGU 694 GAAGCACTT AAGCACTT AGCACTThsa-miR-520d-5p CUACAAAGGGAAGCCCUUUC 695 CCCTTTGTA CCTTTGTA CTTTGTAhsa-miR-520e AAAGUGCUUCCUUUUUGAGGG 696 GAAGCACTT AAGCACTT AGCACTThsa-miR-520f AAGUGCUUCCUUUUAGAGGGUU 697 GGAAGCACT GAAGCACT AAGCACThsa-miR-520g ACAAAGUGCUUCCCUUUAGAGUGU 698 AGCACTTTG GCACTTTG CACTTTGhsa-miR-520h ACAAAGUGCUUCCCUUUAGAGU 699 AGCACTTTG GCACTTTG CACTTTGhsa-miR-521 AACGCACUUCCCUUUAGAGUGU 700 GAAGTGCGT AAGTGCGT AGTGCGThsa-miR-522 AAAAUGGUUCCCUUUAGAGUGU 701 GAACCATTT AACCATTT ACCATTThsa-miR-523 GAACGCGCUUCCCUAUAGAGGGU 702 AAGCGCGTT AGCGCGTT GCGCGTThsa-miR-524-3p GAAGGCGCUUCCCUUUGGAGU 703 AAGCGCCTT AGCGCCTT GCGCCTThsa-miR-524-5p CUACAAAGGGAAGCACUUUCUC 704 CCCTTTGTA CCTTTGTA CTTTGTAhsa-miR-525-3p GAAGGCGCUUCCCUUUAGAGCG 705 AAGCGCCTT AGCGCCTT GCGCCTThsa-miR-525-5p CUCCAGAGGGAUGCACUUUCU 706 CCCTCTGGA CCTCTGGA CTCTGGAhsa-miR-526b CUCUUGAGGGAAGCACUUUCUGU 707 CCCTCAAGA CCTCAAGA CTCAAGAhsa-miR-526b* GAAAGUGCUUCCUUUUAGAGGC 708 AAGCACTTT AGCACTTT GCACTTThsa-miR-527 CUGCAAAGGGAAGCCCUUUC 709 CCCTTTGCA CCTTTGCA CTTTGCAhsa-miR-532-3p CCUCCCACACCCAAGGCUUGCA 710 GTGTGGGAG TGTGGGAG GTGGGAGhsa-miR-532-5p CAUGCCUUGAGUGUAGGACCGU 711 TCAAGGCAT CAAGGCAT AAGGCAThsa-miR-539 GGAGAAAUUAUCCUUGGUGUGU 712 TAATTTCTC AATTTCTC ATTTCTChsa-miR-541 UGGUGGGCACAGAAUCUGGACU 713 GTGCCCACC TGCCCACC GCCCACChsa-miR-541* AAAGGAUUCUGCUGUCGGUCCCACU 714 AGAATCCTT GAATCCTT AATCCTThsa-miR-542-3p UGUGACAGAUUGAUAACUGAAA 715 ATCTGTCAC TCTGTCAC CTGTCAChsa-miR-542-5p UCGGGGAUCAUCAUGUCACGAGA 716 TGATCCCCG GATCCCCG ATCCCCGhsa-miR-543 AAACAUUCGCGGUGCACUUCUU 717 GCGAATGTT CGAATGTT GAATGTThsa-miR-544 AUUCUGCAUUUUUAGCAAGUUC 718 AATGCAGAA ATGCAGAA TGCAGAAhsa-miR-545 UCAGCAAACAUUUAUUGUGUGC 719 TGTTTGCTG GTTTGCTG TTTGCTGhsa-miR-545* UCAGUAAAUGUUUAUUAGAUGA 720 CATTTACTG ATTTACTG TTTACTGhsa-miR-548a-3p CAAAACUGGCAAUUACUUUUGC 721 GCCAGTTTT CCAGTTTT CAGTTTThsa-miR-548a-5p AAAAGUAAUUGCGAGUUUUACC 722 AATTACTTT ATTACTTT TTACTTThsa-miR-548b-3p CAAGAACCUCAGUUGCUUUUGU 723 GAGGTTCTT AGGTTCTT GGTTCTThsa-miR-548b-5p AAAAGUAAUUGUGGUUUUGGCC 724 AATTACTTT ATTACTTT TTACTTThsa-miR-548c-3p CAAAAAUCUCAAUUACUUUUGC 725 GAGATTTTT AGATTTTT GATTTTThsa-miR-548c-5p AAAAGUAAUUGCGGUUUUUGCC 726 AATTACTTT ATTACTTT TTACTTThsa-miR-548d-3p CAAAAACCACAGUUUCUUUUGC 727 GTGGTTTTT TGGTTTTT GGTTTTThsa-miR-548d-5p AAAAGUAAUUGUGGUUUUUGCC 728 AATTACTTT ATTACTTT TTACTTThsa-miR-548e AAAAACUGAGACUACUUUUGCA 729 CTCAGTTTT TCAGTTTT CAGTTTThsa-miR-548f AAAAACUGUAAUUACUUUU 730 TACAGTTTT ACAGTTTT CAGTTTThsa-miR-548g AAAACUGUAAUUACUUUUGUAC 731 TTACAGTTT TACAGTTT ACAGTTThsa-miR-548h AAAAGUAAUCGCGGUUUUUGUC 732 GATTACTTT ATTACTTT TTACTTThsa-miR-548i AAAAGUAAUUGCGGAUUUUGCC 733 AATTACTTT ATTACTTT TTACTTThsa-miR-548j AAAAGUAAUUGCGGUCUUUGGU 734 AATTACTTT ATTACTTT TTACTTThsa-miR-548k AAAAGUACUUGCGGAUUUUGCU 735 AAGTACTTT AGTACTTT GTACTTThsa-miR-548l AAAAGUAUUUGCGGGUUUUGUC 736 AAATACTTT AATACTTT ATACTTThsa-miR-548m CAAAGGUAUUUGUGGUUUUUG 737 AATACCTTT ATACCTTT TACCTTThsa-miR-548n CAAAAGUAAUUGUGGAUUUUGU 738 ATTACTTTT TTACTTTT TACTTTThsa-miR-548o CCAAAACUGCAGUUACUUUUGC 739 GCAGTTTTG CAGTTTTG AGTTTTGhsa-miR-548p UAGCAAAAACUGCAGUUACUUU 740 GTTTTTGCT TTTTTGCT TTTTGCThsa-miR-549 UGACAACUAUGGAUGAGCUCU 741 ATAGTTGTC TAGTTGTC AGTTGTChsa-miR-550 AGUGCCUGAGGGAGUAAGAGCCC 742 CTCAGGCAC TCAGGCAC CAGGCAChsa-miR-550* UGUCUUACUCCCUCAGGCACAU 743 GAGTAAGAC AGTAAGAC GTAAGAChsa-miR-551a GCGACCCACUCUUGGUUUCCA 744 AGTGGGTCG GTGGGTCG TGGGTCGhsa-miR-551b GCGACCCAUACUUGGUUUCAG 745 TATGGGTCG ATGGGTCG TGGGTCGhsa-miR-551b* GAAAUCAAGCGUGGGUGAGACC 746 GCTTGATTT CTTGATTT TTGATTThsa-miR-552 AACAGGUGACUGGUUAGACAA 747 GTCACCTGT TCACCTGT CACCTGThsa-miR-553 AAAACGGUGAGAUUUUGUUUU 748 TCACCGTTT CACCGTTT ACCGTTThsa-miR-554 GCUAGUCCUGACUCAGCCAGU 749 CAGGACTAG AGGACTAG GGACTAGhsa-miR-555 AGGGUAAGCUGAACCUCUGAU 750 AGCTTACCC GCTTACCC CTTACCChsa-miR-556-3p AUAUUACCAUUAGCUCAUCUUU 751 ATGGTAATA TGGTAATA GGTAATAhsa-miR-556-5p GAUGAGCUCAUUGUAAUAUGAG 752 TGAGCTCAT GAGCTCAT AGCTCAThsa-miR-557 GUUUGCACGGGUGGGCCUUGUCU 753 CCGTGCAAA CGTGCAAA GTGCAAAhsa-miR-558 UGAGCUGCUGUACCAAAAU 754 CAGCAGCTC AGCAGCTC GCAGCTChsa-miR-559 UAAAGUAAAUAUGCACCAAAA 755 ATTTACTTT TTTACTTT TTACTTThsa-miR-561 CAAAGUUUAAGAUCCUUGAAGU 756 TTAAACTTT TAAACTTT AAACTTThsa-miR-562 AAAGUAGCUGUACCAUUUGC 757 CAGCTACTT AGCTACTT GCTACTThsa-miR-563 AGGUUGACAUACGUUUCCC 758 ATGTCAACC TGTCAACC GTCAACChsa-miR-564 AGGCACGGUGUCAGCAGGC 759 CACCGTGCC ACCGTGCC CCGTGCChsa-miR-566 GGGCGCCUGUGAUCCCAAC 760 ACAGGCGCC CAGGCGCC AGGCGCChsa-miR-567 AGUAUGUUCUUCCAGGACAGAAC 761 AGAACATAC GAACATAC AACATAChsa-miR-568 AUGUAUAAAUGUAUACACAC 762 ATTTATACA TTTATACA TTATACAhsa-miR-569 AGUUAAUGAAUCCUGGAAAGU 763 TTCATTAAC TCATTAAC CATTAAChsa-miR-570 CGAAAACAGCAAUUACCUUUGC 764 GCTGTTTTC CTGTTTTC TGTTTTChsa-miR-571 UGAGUUGGCCAUCUGAGUGAG 765 GGCCAACTC GCCAACTC CCAACTChsa-miR-572 GUCCGCUCGGCGGUGGCCCA 766 CCGAGCGGA CGAGCGGA GAGCGGAhsa-miR-573 CUGAAGUGAUGUGUAACUGAUCAG 767 ATCACTTCA TCACTTCA CACTTCAhsa-miR-574-3p CACGCUCAUGCACACACCCACA 768 CATGAGCGT ATGAGCGT TGAGCGThsa-miR-574-5p UGAGUGUGUGUGUGUGAGUGUGU 769 CACACACTC ACACACTC CACACTChsa-miR-575 GAGCCAGUUGGACAGGAGC 770 CAACTGGCT AACTGGCT ACTGGCThsa-miR-576-3p AAGAUGUGGAAAAAUUGGAAUC 771 TCCACATCT CCACATCT CACATCThsa-miR-576-5p AUUCUAAUUUCUCCACGUCUUU 772 AAATTAGAA AATTAGAA ATTAGAAhsa-miR-577 UAGAUAAAAUAUUGGUACCUG 773 ATTTTATCT TTTTATCT TTTATCThsa-miR-578 CUUCUUGUGCUCUAGGAUUGU 774 GCACAAGAA CACAAGAA ACAAGAAhsa-miR-579 UUCAUUUGGUAUAAACCGCGAUU 775 ACCAAATGA CCAAATGA CAAATGAhsa-miR-580 UUGAGAAUGAUGAAUCAUUAGG 776 TCATTCTCA CATTCTCA ATTCTCAhsa-miR-581 UCUUGUGUUCUCUAGAUCAGU 777 GAACACAAG AACACAAG ACACAAGhsa-miR-582-3p UAACUGGUUGAACAACUGAACC 778 CAACCAGTT AACCAGTT ACCAGTThsa-miR-582-5p UUACAGUUGUUCAACCAGUUACU 779 ACAACTGTA CAACTGTA AACTGTAhsa-miR-583 CAAAGAGGAAGGUCCCAUUAC 780 TTCCTCTTT TCCTCTTT CCTCTTThsa-miR-584 UUAUGGUUUGCCUGGGACUGAG 781 CAAACCATA AAACCATA AACCATAhsa-miR-585 UGGGCGUAUCUGUAUGCUA 782 GATACGCCC ATACGCCC TACGCCChsa-miR-586 UAUGCAUUGUAUUUUUAGGUCC 783 ACAATGCAT CAATGCAT AATGCAThsa-miR-587 UUUCCAUAGGUGAUGAGUCAC 784 CCTATGGAA CTATGGAA TATGGAAhsa-miR-588 UUGGCCACAAUGGGUUAGAAC 785 TTGTGGCCA TGTGGCCA GTGGCCAhsa-miR-589 UGAGAACCACGUCUGCUCUGAG 786 GTGGTTCTC TGGTTCTC GGTTCTChsa-miR-589* UCAGAACAAAUGCCGGUUCCCAGA 787 TTTGTTCTG TTGTTCTG TGTTCTGhsa-miR-590-3p UAAUUUUAUGUAUAAGCUAGU 788 CATAAAATT ATAAAATT TAAAATThsa-miR-590-5p GAGCUUAUUCAUAAAAGUGCAG 789 GAATAAGCT AATAAGCT ATAAGCThsa-miR-591 AGACCAUGGGUUCUCAUUGU 790 CCCATGGTC CCATGGTC CATGGTChsa-miR-592 UUGUGUCAAUAUGCGAUGAUGU 791 ATTGACACA TTGACACA TGACACAhsa-miR-593 UGUCUCUGCUGGGGUUUCU 792 AGCAGAGAC GCAGAGAC CAGAGAChsa-miR-593* AGGCACCAGCCAGGCAUUGCUCAGC 793 GCTGGTGCC CTGGTGCC TGGTGCChsa-miR-595 GAAGUGUGCCGUGGUGUGUCU 794 GGCACACTT GCACACTT CACACTThsa-miR-596 AAGCCUGCCCGGCUCCUCGGG 795 GGGCAGGCT GGCAGGCT GCAGGCThsa-miR-597 UGUGUCACUCGAUGACCACUGU 796 GAGTGACAC AGTGACAC GTGACAChsa-miR-598 UACGUCAUCGUUGUCAUCGUCA 797 CGATGACGT GATGACGT ATGACGThsa-miR-599 GUUGUGUCAGUUUAUCAAAC 798 CTGACACAA TGACACAA GACACAAhsa-miR-600 ACUUACAGACAAGAGCCUUGCUC 799 GTCTGTAAG TCTGTAAG CTGTAAGhsa-miR-601 UGGUCUAGGAUUGUUGGAGGAG 800 TCCTAGACC CCTAGACC CTAGACChsa-miR-602 GACACGGGCGACAGCUGCGGCCC 801 CGCCCGTGT GCCCGTGT CCCGTGThsa-miR-603 CACACACUGCAAUUACUUUUGC 802 GCAGTGTGT CAGTGTGT AGTGTGThsa-miR-604 AGGCUGCGGAAUUCAGGAC 803 TCCGCAGCC CCGCAGCC CGCAGCChsa-miR-605 UAAAUCCCAUGGUGCCUUCUCCU 804 ATGGGATTT TGGGATTT GGGATTThsa-miR-606 AAACUACUGAAAAUCAAAGAU 805 TCAGTAGTT CAGTAGTT AGTAGTThsa-miR-607 GUUCAAAUCCAGAUCUAUAAC 806 GGATTTGAA GATTTGAA ATTTGAAhsa-miR-608 AGGGGUGGUGUUGGGACAGCUCCGU 807 CACCACCCC ACCACCCC CCACCCChsa-miR-609 AGGGUGUUUCUCUCAUCUCU 808 GAAACACCC AAACACCC AACACCChsa-miR-610 UGAGCUAAAUGUGUGCUGGGA 809 ATTTAGCTC TTTAGCTC TTAGCTChsa-miR-611 GCGAGGACCCCUCGGGGUCUGAC 810 GGGTCCTCG GGTCCTCG GTCCTCGhsa-miR-612 GCUGGGCAGGGCUUCUGAGCUCCUU 811 CCTGCCCAG CTGCCCAG TGCCCAGhsa-miR-613 AGGAAUGUUCCUUCUUUGCC 812 GAACATTCC AACATTCC ACATTCChsa-miR-614 GAACGCCUGUUCUUGCCAGGUGG 813 ACAGGCGTT CAGGCGTT AGGCGTThsa-miR-615-3p UCCGAGCCUGGGUCUCCCUCUU 814 CAGGCTCGG AGGCTCGG GGCTCGGhsa-miR-615-5p GGGGGUCCCCGGUGCUCGGAUC 815 GGGGACCCC GGGACCCC GGACCCChsa-miR-616 AGUCAUUGGAGGGUUUGAGCAG 816 TCCAATGAC CCAATGAC CAATGAChsa-miR-616* ACUCAAAACCCUUCAGUGACUU 817 GGTTTTGAG GTTTTGAG TTTTGAGhsa-miR-617 AGACUUCCCAUUUGAAGGUGGC 818 TGGGAAGTC GGGAAGTC GGAAGTChsa-miR-618 AAACUCUACUUGUCCUUCUGAGU 819 AGTAGAGTT GTAGAGTT TAGAGTThsa-miR-619 GACCUGGACAUGUUUGUGCCCAGU 820 TGTCCAGGT GTCCAGGT TCCAGGThsa-miR-620 AUGGAGAUAGAUAUAGAAAU 821 CTATCTCCA TATCTCCA ATCTCCAhsa-miR-621 GGCUAGCAACAGCGCUUACCU 822 GTTGCTAGC TTGCTAGC TGCTAGChsa-miR-622 ACAGUCUGCUGAGGUUGGAGC 823 AGCAGACTG GCAGACTG CAGACTGhsa-miR-623 AUCCCUUGCAGGGGCUGUUGGGU 824 TGCAAGGGA GCAAGGGA CAAGGGAhsa-miR-624 CACAAGGUAUUGGUAUUACCU 825 ATACCTTGT TACCTTGT ACCTTGThsa-miR-624* UAGUACCAGUACCUUGUGUUCA 826 ACTGGTACT CTGGTACT TGGTACThsa-miR-625 AGGGGGAAAGUUCUAUAGUCC 827 CTTTCCCCC TTTCCCCC TTCCCCChsa-miR-625* GACUAUAGAACUUUCCCCCUCA 828 TTCTATAGT TCTATAGT CTATAGThsa-miR-626 AGCUGUCUGAAAAUGUCUU 829 TCAGACAGC CAGACAGC AGACAGChsa-miR-627 GUGAGUCUCUAAGAAAAGAGGA 830 AGAGACTCA GAGACTCA AGACTCAhsa-miR-628-3p UCUAGUAAGAGUGGCAGUCGA 831 TCTTACTAG CTTACTAG TTACTAGhsa-miR-628-5p AUGCUGACAUAUUUACUAGAGG 832 ATGTCAGCA TGTCAGCA GTCAGCAhsa-miR-629 UGGGUUUACGUUGGGAGAACU 833 CGTAAACCC GTAAACCC TAAACCChsa-miR-629* GUUCUCCCAACGUAAGCCCAGC 834 TTGGGAGAA TGGGAGAA GGGAGAAhsa-miR-630 AGUAUUCUGUACCAGGGAAGGU 835 ACAGAATAC CAGAATAC AGAATAChsa-miR-631 AGACCUGGCCCAGACCUCAGC 836 GGCCAGGTC GCCAGGTC CCAGGTChsa-miR-632 GUGUCUGCUUCCUGUGGGA 837 AAGCAGACA AGCAGACA GCAGACAhsa-miR-633 CUAAUAGUAUCUACCACAAUAAA 838 ATACTATTA TACTATTA ACTATTAhsa-miR-634 AACCAGCACCCCAACUUUGGAC 839 GGTGCTGGT GTGCTGGT TGCTGGThsa-miR-635 ACUUGGGCACUGAAACAAUGUCC 840 GTGCCCAAG TGCCCAAG GCCCAAGhsa-miR-636 UGUGCUUGCUCGUCCCGCCCGCA 841 AGCAAGCAC GCAAGCAC CAAGCAChsa-miR-637 ACUGGGGGCUUUCGGGCUCUGCGU 842 AGCCCCCAG GCCCCCAG CCCCCAGhsa-miR-638 AGGGAUCGCGGGCGGGUGGCGGCCU 843 CGCGATCCC GCGATCCC CGATCCChsa-miR-639 AUCGCUGCGGUUGCGAGCGCUGU 844 CCGCAGCGA CGCAGCGA GCAGCGAhsa-miR-640 AUGAUCCAGGAACCUGCCUCU 845 CCTGGATCA CTGGATCA TGGATCAhsa-miR-641 AAAGACAUAGGAUAGAGUCACCUC 846 CTATGTCTT TATGTCTT ATGTCTThsa-miR-642 GUCCCUCUCCAAAUGUGUCUUG 847 GGAGAGGGA GAGAGGGA AGAGGGAhsa-miR-643 ACUUGUAUGCUAGCUCAGGUAG 848 GCATACAAG CATACAAG ATACAAGhsa-miR-644 AGUGUGGCUUUCUUAGAGC 849 AAGCCACAC AGCCACAC GCCACAChsa-miR-645 UCUAGGCUGGUACUGCUGA 850 CCAGCCTAG CAGCCTAG AGCCTAGhsa-miR-646 AAGCAGCUGCCUCUGAGGC 851 GCAGCTGCT CAGCTGCT AGCTGCThsa-miR-647 GUGGCUGCACUCACUUCCUUC 852 GTGCAGCCA TGCAGCCA GCAGCCAhsa-miR-648 AAGUGUGCAGGGCACUGGU 853 CTGCACACT TGCACACT GCACACThsa-miR-649 AAACCUGUGUUGUUCAAGAGUC 854 ACACAGGTT CACAGGTT ACAGGTThsa-miR-650 AGGAGGCAGCGCUCUCAGGAC 855 GCTGCCTCC CTGCCTCC TGCCTCChsa-miR-651 UUUAGGAUAAGCUUGACUUUUG 856 TTATCCTAA TATCCTAA ATCCTAAhsa-miR-652 AAUGGCGCCACUAGGGUUGUG 857 TGGCGCCAT GGCGCCAT GCGCCAThsa-miR-653 GUGUUGAAACAAUCUCUACUG 858 GTTTCAACA TTTCAACA TTCAACAhsa-miR-654-3p UAUGUCUGCUGACCAUCACCUU 859 AGCAGACAT GCAGACAT CAGACAThsa-miR-654-5p UGGUGGGCCGCAGAACAUGUGC 860 CGGCCCACC GGCCCACC GCCCACChsa-miR-655 AUAAUACAUGGUUAACCUCUUU 861 CATGTATTA ATGTATTA TGTATTAhsa-miR-656 AAUAUUAUACAGUCAACCUCU 862 GTATAATAT TATAATAT ATAATAThsa-miR-657 GGCAGGUUCUCACCCUCUCUAGG 863 AGAACCTGC GAACCTGC AACCTGChsa-miR-658 GGCGGAGGGAAGUAGGUCCGUUGGU 864 TCCCTCCGC CCCTCCGC CCTCCGChsa-miR-659 CUUGGUUCAGGGAGGGUCCCCA 865 CTGAACCAA TGAACCAA GAACCAAhsa-miR-660 UACCCAUUGCAUAUCGGAGUUG 866 GCAATGGGT CAATGGGT AATGGGThsa-miR-661 UGCCUGGGUCUCUGGCCUGCGCGU 867 GACCCAGGC ACCCAGGC CCCAGGChsa-miR-662 UCCCACGUUGUGGCCCAGCAG 868 CAACGTGGG AACGTGGG ACGTGGGhsa-miR-663 AGGCGGGGCGCCGCGGGACCGC 869 CGCCCCGCC GCCCCGCC CCCCGCChsa-miR-663b GGUGGCCCGGCCGUGCCUGAGG 870 CCGGGCCAC CGGGCCAC GGGCCAChsa-miR-664 UAUUCAUUUAUCCCCAGCCUACA 871 TAAATGAAT AAATGAAT AATGAAThsa-miR-664* ACUGGCUAGGGAAAAUGAUUGGAU 872 CCTAGCCAG CTAGCCAG TAGCCAGhsa-miR-665 ACCAGGAGGCUGAGGCCCCU 873 GCCTCCTGG CCTCCTGG CTCCTGGhsa-miR-668 UGUCACUCGGCUCGGCCCACUAC 874 CCGAGTGAC CGAGTGAC GAGTGAChsa-miR-671-3p UCCGGUUCUCAGGGCUCCACC 875 GAGAACCGG AGAACCGG GAACCGGhsa-miR-671-5p AGGAAGCCCUGGAGGGGCUGGAG 876 AGGGCTTCC GGGCTTCC GGCTTCChsa-miR-675 UGGUGCGGAGAGGGCCCACAGUG 877 CTCCGCACC TCCGCACC CCGCACChsa-miR-675b CUGUAUGCCCUCACCGCUCA 878 GGGCATACA GGCATACA GCATACAhsa-miR-7 UGGAAGACUAGUGAUUUUGUUGU 879 TAGTCTTCC AGTCTTCC GTCTTCChsa-miR-7-1* CAACAAAUCACAGUCUGCCAUA 880 TGATTTGTT GATTTGTT ATTTGTThsa-miR-7-2* CAACAAAUCCCAGUCUACCUAA 881 GGATTTGTT GATTTGTT ATTTGTThsa-miR-708 AAGGAGCUUACAAUCUAGCUGGG 882 TAAGCTCCT AAGCTCCT AGCTCCThsa-miR-708* CAACUAGACUGUGAGCUUCUAG 883 AGTCTAGTT GTCTAGTT TCTAGTThsa-miR-720 UCUCGCUGGGGCCUCCA 884 CCCAGCGAG CCAGCGAG CAGCGAG hsa-miR-744UGCGGGGCUAGGGCUAACAGCA 885 TAGCCCCGC AGCCCCGC GCCCCGC hsa-miR-744*CUGUUGCCACUAACCUCAACCU 886 GTGGCAACA TGGCAACA GGCAACA hsa-miR-758UUUGUGACCUGGUCCACUAACC 887 AGGTCACAA GGTCACAA GTCACAA hsa-miR-760CGGCUCUGGGUCUGUGGGGA 888 CCCAGAGCC CCAGAGCC CAGAGCC hsa-miR-765UGGAGGAGAAGGAAGGUGAUG 889 TTCTCCTCC TCTCCTCC CTCCTCC hsa-miR-766ACUCCAGCCCCACAGCCUCAGC 890 GGGCTGGAG GGCTGGAG GCTGGAG hsa-miR-767-3pUCUGCUCAUACCCCAUGGUUUCU 891 TATGAGCAG ATGAGCAG TGAGCAG hsa-miR-767-5pUGCACCAUGGUUGUCUGAGCAUG 892 CCATGGTGC CATGGTGC ATGGTGC hsa-miR-769-3pCUGGGAUCUCCGGGGUCUUGGUU 893 GAGATCCCA AGATCCCA GATCCCA hsa-miR-769-5pUGAGACCUCUGGGUUCUGAGCU 894 AGAGGTCTC GAGGTCTC AGGTCTC hsa-miR-770-5pUCCAGUACCACGUGUCAGGGCCA 895 TGGTACTGG GGTACTGG GTACTGG hsa-miR-802CAGUAACAAAGAUUCAUCCUUGU 896 TTTGTTACT TTGTTACT TGTTACT hsa-miR-873GCAGGAACUUGUGAGUCUCCU 897 AAGTTCCTG AGTTCCTG GTTCCTG hsa-miR-874CUGCCCUGGCCCGAGGGACCGA 898 GCCAGGGCA CCAGGGCA CAGGGCA hsa-miR-875-3pCCUGGAAACACUGAGGUUGUG 899 TGTTTCCAG GTTTCCAG TTTCCAG hsa-miR-875-5pUAUACCUCAGUUUUAUCAGGUG 900 CTGAGGTAT TGAGGTAT GAGGTAT hsa-miR-876-3pUGGUGGUUUACAAAGUAAUUCA 901 TAAACCACC AAACCACC AACCACC hsa-miR-876-5pUGGAUUUCUUUGUGAAUCACCA 902 AAGAAATCC AGAAATCC GAAATCC hsa-miR-877GUAGAGGAGAUGGCGCAGGG 903 TCTCCTCTA CTCCTCTA TCCTCTA hsa-miR-877*UCCUCUUCUCCCUCCUCCCAG 904 GAGAAGAGG AGAAGAGG GAAGAGG hsa-miR-885-3pAGGCAGCGGGGUGUAGUGGAUA 905 CCCGCTGCC CCGCTGCC CGCTGCC hsa-miR-885-5pUCCAUUACACUACCCUGCCUCU 906 GTGTAATGG TGTAATGG GTAATGG hsa-miR-886-3pCGCGGGUGCUUACUGACCCUU 907 AGCACCCGC GCACCCGC CACCCGC hsa-miR-886-5pCGGGUCGGAGUUAGCUCAAGCGG 908 CTCCGACCC TCCGACCC CCGACCC hsa-miR-887GUGAACGGGCGCCAUCCCGAGG 909 GCCCGTTCA CCCGTTCA CCGTTCA hsa-miR-888UACUCAAAAAGCUGUCAGUCA 910 TTTTTGAGT TTTTGAGT TTTGAGT hsa-miR-888*GACUGACACCUCUUUGGGUGAA 911 GGTGTCAGT GTGTCAGT TGTCAGT hsa-miR-889UUAAUAUCGGACAACCAUUGU 912 CCGATATTA CGATATTA GATATTA hsa-miR-890UACUUGGAAAGGCAUCAGUUG 913 TTTCCAAGT TTCCAAGT TCCAAGT hsa-miR-891aUGCAACGAACCUGAGCCACUGA 914 GTTCGTTGC TTCGTTGC TCGTTGC hsa-miR-891bUGCAACUUACCUGAGUCAUUGA 915 GTAAGTTGC TAAGTTGC AAGTTGC hsa-miR-892aCACUGUGUCCUUUCUGCGUAG 916 GGACACAGT GACACAGT ACACAGT hsa-miR-892bCACUGGCUCCUUUCUGGGUAGA 917 GGAGCCAGT GAGCCAGT AGCCAGT hsa-miR-9UCUUUGGUUAUCUAGCUGUAUGA 918 TAACCAAAG AACCAAAG ACCAAAG hsa-miR-9*AUAAAGCUAGAUAACCGAAAGU 919 CTAGCTTTA TAGCTTTA AGCTTTA hsa-miR-920GGGGAGCUGUGGAAGCAGUA 920 ACAGCTCCC CAGCTCCC AGCTCCC hsa-miR-921CUAGUGAGGGACAGAACCAGGAUUC 921 CCCTCACTA CCTCACTA CTCACTA hsa-miR-922GCAGCAGAGAAUAGGACUACGUC 922 TCTCTGCTG CTCTGCTG TCTGCTG hsa-miR-923GUCAGCGGAGGAAAAGAAACU 923 CTCCGCTGA TCCGCTGA CCGCTGA hsa-miR-924AGAGUCUUGUGAUGUCUUGC 924 ACAAGACTC CAAGACTC AAGACTC hsa-miR-92aUAUUGCACUUGUCCCGGCCUGU 925 AAGTGCAAT AGTGCAAT GTGCAAT hsa-miR-92a-1*AGGUUGGGAUCGGUUGCAAUGCU 926 ATCCCAACC TCCCAACC CCCAACC hsa-miR-92a-2*GGGUGGGGAUUUGUUGCAUUAC 927 ATCCCCACC TCCCCACC CCCCACC hsa-miR-92bUAUUGCACUCGUCCCGGCCUCC 928 GAGTGCAAT AGTGCAAT GTGCAAT hsa-miR-92b*AGGGACGGGACGCGGUGCAGUG 929 TCCCGTCCC CCCGTCCC CCGTCCC hsa-miR-93CAAAGUGCUGUUCGUGCAGGUAG 930 CAGCACTTT AGCACTTT GCACTTT hsa-miR-93*ACUGCUGAGCUAGCACUUCCCG 931 GCTCAGCAG CTCAGCAG TCAGCAG hsa-miR-933UGUGCGCAGGGAGACCUCUCCC 932 CCTGCGCAC CTGCGCAC TGCGCAC hsa-miR-934UGUCUACUACUGGAGACACUGG 933 GTAGTAGAC TAGTAGAC AGTAGAC hsa-miR-935CCAGUUACCGCUUCCGCUACCGC 934 CGGTAACTG GGTAACTG GTAACTG hsa-miR-936ACAGUAGAGGGAGGAAUCGCAG 935 CCTCTACTG CTCTACTG TCTACTG hsa-miR-937AUCCGCGCUCUGACUCUCUGCC 936 GAGCGCGGA AGCGCGGA GCGCGGA hsa-miR-938UGCCCUUAAAGGUGAACCCAGU 937 TTTAAGGGC TTAAGGGC TAAGGGC hsa-miR-939UGGGGAGCUGAGGCUCUGGGGGUG 938 CAGCTCCCC AGCTCCCC GCTCCCC hsa-miR-940AAGGCAGGGCCCCCGCUCCCC 939 GCCCTGCCT CCCTGCCT CCTGCCT hsa-miR-941CACCCGGCUGUGUGCACAUGUGC 940 CAGCCGGGT AGCCGGGT GCCGGGT hsa-miR-942UCUUCUCUGUUUUGGCCAUGUG 941 ACAGAGAAG CAGAGAAG AGAGAAG hsa-miR-943CUGACUGUUGCCGUCCUCCAG 942 CAACAGTCA AACAGTCA ACAGTCA hsa-miR-944AAAUUAUUGUACAUCGGAUGAG 943 ACAATAATT CAATAATT AATAATT hsa-miR-95UUCAACGGGUAUUUAUUGAGCA 944 ACCCGTTGA CCCGTTGA CCGTTGA hsa-miR-96UUUGGCACUAGCACAUUUUUGCU 945 TAGTGCCAA AGTGCCAA GTGCCAA hsa-miR-96*AAUCAUGUGCAGUGCCAAUAUG 946 GCACATGAT CACATGAT ACATGAT hsa-miR-98UGAGGUAGUAAGUUGUAUUGUU 947 TACTACCTC ACTACCTC CTACCTC hsa-miR-99aAACCCGUAGAUCCGAUCUUGUG 948 TCTACGGGT CTACGGGT TACGGGT hsa-miR-99a*CAAGCUCGCUUCUAUGGGUCUG 949 AGCGAGCTT GCGAGCTT CGAGCTT hsa-miR-99bCACCCGUAGAACCGACCUUGCG 950 TCTACGGGT CTACGGGT TACGGGT hsa-miR-99b*CAAGCUCGUGUCUGUGGGUCCG 951 CACGAGCTT ACGAGCTT CGAGCTT hsvl-miR-H1UGGAAGGACGGGAAGUGGAAG 952 CGTCCTTCC GTCCTTCC TCCTTCC hsvl-miR-H2-3pCCUGAGCCAGGGACGAGUGCGACU 953 CTGGCTCAG TGGCTCAG GGCTCAG hsvl-miR-H2-5pUCGCACGCGCCCGGCACAGACU 954 GCGCGTGCG CGCGTGCG GCGTGCG hsvl-miR-H3CUGGGACUGUGCGGUUGGGA 955 ACAGTCCCA CAGTCCCA AGTCCCA hsvl-miR-H4-3pCUUGCCUGUCUAACUCGCUAGU 956 GACAGGCAA ACAGGCAA CAGGCAA hsvl-miR-H4-5pGGUAGAGUUUGACAGGCAAGCA 957 AAACTCTAC AACTCTAC ACTCTAC hsvl-miR-H5GUCAGAGAUCCAAACCCUCCGG 958 GATCTCTGA ATCTCTGA TCTCTGA hsvl-miR-H6CACUUCCCGUCCUUCCAUCCC 959 ACGGGAAGT CGGGAAGT GGGAAGT kshv-miR-K12-1AUUACAGGAAACUGGGUGUAAGC 960 TTCCTGTAA TCCTGTAA CCTGTAA kshv-miR-K12-10aUAGUGUUGUCCCCCCGAGUGGC 961 GACAACACT ACAACACT CAACACT kshv-miR-K12-10bUGGUGUUGUCCCCCCGAGUGGC 962 GACAACACC ACAACACC CAACACC kshv-miR-K12-11UUAAUGCUUAGCCUGUGUCCGA 963 TAAGCATTA AAGCATTA AGCATTA kshv-miR-K12-12ACCAGGCCACCAUUCCUCUCCG 964 GTGGCCTGG TGGCCTGG GGCCTGG kshv-miR-K12-2AACUGUAGUCCGGGUCGAUCUG 965 GACTACAGT ACTACAGT CTACAGT kshv-miR-K12-3UCACAUUCUGAGGACGGCAGCGA 966 CAGAATGTG AGAATGTG GAATGTG kshv-miR-K12-3*UCGCGGUCACAGAAUGUGACA 967 GTGACCGCG TGACCGCG GACCGCG kshv-miR-K12-4-UAGAAUACUGAGGCCUAGCUGA 968 CAGTATTCT AGTATTCT GTATTCT 3p kshv-miR-K12-4-AGCUAAACCGCAGUACUCUAGG 969 CGGTTTAGC GGTTTAGC GTTTAGC 5p kshv-miR-K12-5UAGGAUGCCUGGAACUUGCCGG 970 AGGCATCCT GGCATCCT GCATCCT kshv-miR-K12-6-UGAUGGUUUUCGGGCUGUUGAG 971 AAAACCATC AAACCATC AACCATC 3p kshv-miR-K12-6-CCAGCAGCACCUAAUCCAUCGG 972 GTGCTGCTG TGCTGCTG GCTGCTG 5p kshv-miR-K12-7UGAUCCCAUGUUGCUGGCGCU 973 CATGGGATC ATGGGATC TGGGATC kshv-miR-K12-8UAGGCGCGACUGAGAGAGCACG 974 GTCGCGCCT TCGCGCCT CGCGCCT kshv-miR-K12-9CUGGGUAUACGCAGCUGCGUAA 975 GTATACCCA TATACCCA ATACCCA kshv-miR-K12-9*ACCCAGCUGCGUAAACCCCGCU 976 GCAGCTGGG CAGCTGGG AGCTGGG

1-34. (canceled)
 35. An oligomer of a contiguous sequence of 7, 8, 9, or10 nucleotide units in length, for use in reducing the effective amountof a microRNA target in a cell or an organism, wherein at least 70% ofthe nucleotide units of the oligomer are selected from the groupconsisting of LNA (Locked Nucleic Acid) units and 2′ substitutednucleotide analogues, and wherein at least 50% of the nucleotide unitsof the oligomer are LNA units, and wherein at least one of theinternucleoside linkages present between the nucleotide units of thecontiguous nucleotide sequence is a phosphorothioate internucleosidelinkage, wherein the contiguous nucleotide sequence is 100%complementary to the seed sequence of miR-155 (SEQ ID NO: 315), andwherein the seed sequence of miR-155 consists of nucleotides 2 to 8 ofSEQ ID NO: 315) counting from the sequence's 5′ end.
 36. The oligomeraccording to claim 35, for use in the treatment of a medical disorder ordisease wherein the medical disease or disorder is lymphoma, pancreaticcancer, breast cancer, or lung cancer.
 37. A pharmaceutical compositioncomprising the oligomer according to claim 35, and a pharmaceuticallyacceptable diluent, carrier, salt or adjuvant.
 38. The pharmaceuticalcomposition according to claim 37, further comprising a secondindependent active ingredient that is a chemotherapeutic agent.
 39. Amethod for the treatment of a disease or medical disorder associatedwith the presence or over-expression of a microRNA, comprising the stepof administering the pharmaceutical composition according to claim 37 toa patient who is suffering from, or is likely to suffer from saiddisease or medical disorder.
 40. The oligomer according to claim 35,wherein at least 75% of the internucleoside linkages present between thenucleotide units of the contiguous nucleotide sequence arephosphorothioate internucleoside linkages.
 41. The oligomer according toclaim 35, wherein all the internucleoside linkages present between thenucleotide units of the contiguous nucleotide sequence arephosphorothioate internucleoside linkages.
 42. The oligomer according toclaim 40, wherein the nucleotide units are selected from the groupconsisting of DNA, 2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNAunit, 2′-fluoro-DNA unit, LNA unit, PNA unit, HNA unit, INA unit, and a2′-MOE RNA unit.
 43. The oligomer according to claim 40 which comprisesa 3′ terminal LNA unit and a 5′ terminal LNA unit.
 44. The oligomeraccording to claim 40, wherein the length of the oligomer is 7, 8 or 9contiguous nucleotides, wherein each one of the contiguous nucleotidesindependently consists of a LNA unit or a 2′ substituted nucleotideanalogue unit, and wherein none of the contiguous nucleotides is a DNAunit.
 45. The oligomer according to claim 41, wherein all of thenucleotide units of the contiguous nucleotide sequence are LNA units.46. The oligomer according to claim 35, wherein the contiguousnucleotide sequence of the oligomer is 7 nucleotide units in length, allthe nucleotide units are LNA units, and all the internucleoside linkagesare phosphorothioate.
 47. The oligomer according to claim 35, whereinthe contiguous nucleotide sequence of the oligomer is 8 nucleotide unitsin length, all the nucleotide units are LNA units, and all theinternucleoside linkages are phosphorothioate.
 48. The oligomeraccording to claim 35, wherein the contiguous nucleotide sequence of theoligomer is 9 nucleotide units in length, all the nucleotide units areLNA units, and all the internucleoside linkages are phosphorothioate.49. The oligomer according to claim 45, wherein said contiguousnucleotide sequence of the oligomer does not comprise a nucleotide whichcorresponds to the first nucleotide present in the microRNA sequence ofmiR-155 (SEQ ID NO:315) counted from the 5′ end.
 50. The oligomeraccording to claim 35, wherein the contiguous nucleotide sequence of theoligomer comprises the sequence: (a) 5′-TTAGCATTA-3′; (SEQ ID NO: 986)(b) 5′-TAGCATTA-3′; (SEQ ID NO: 4) or, (c) 5′-AGCATTA-3′. (SEQ ID NO:987)


51. The oligomer according to claim 40, wherein the contiguousnucleotide sequence of the oligomer comprises the sequence: (a)5′-TTAGCATTA-3′; (SEQ ID NO: 986) (b) 5′-TAGCATTA-3′; (SEQ ID NO: 4) or,(c) 5′-AGCATTA-3′. (SEQ ID NO: 987)


52. The oligomer according to claim 41, wherein the contiguousnucleotide sequence of the oligomer comprises the sequence: (a)5′-TTAGCATTA-3′; (SEQ ID NO: 986) (b) 5′-TAGCATTA-3′; (SEQ ID NO: 4) or,(c) 5′-AGCATTA-3′. (SEQ ID NO: 987)


53. The oligomer according to claim 36, wherein the contiguousnucleotide sequence of the oligomer is: (a) 5′-TTAGCATTA-3′; (SEQ ID NO:986) (b) 5′-TAGCATTA-3′; (SEQ ID NO: 4) or, (c) 5′-AGCATTA-3′. (SEQ IDNO: 987)


54. The oligomer according to claim 40, wherein the contiguousnucleotide sequence of the oligomer is: (a) 5′-TTAGCATTA-3′; (SEQ ID NO:986) (b) 5′-TAGCATTA-3′; (SEQ ID NO: 4) or, (c) 5′-AGCATTA-3′. (SEQ IDNO: 987)


55. The oligomer according to claim 41, wherein the contiguousnucleotide sequence of the oligomer is: (a) 5′-TTAGCATTA-3′; (SEQ ID NO:986) (b) 5′-TAGCATTA-3′; (SEQ ID NO: 4) or, (c) 5′-AGCATTA-3′. (SEQ IDNO: 987)


56. The oligomer according to claim 45, wherein the contiguousnucleotide sequence of the oligomer is 7 nucleotide units in length, allthe nucleotide units are LNA units, all the internucleoside linkages arephosphorothioate, and all cytosine LNA units are 5-methylcytosines. 57.The oligomer according to claim 56, wherein the oligomer sequencecomprises 5′-AGCATTA-3′ (SEQ ID NO: 987).
 58. The oligomer according toclaim 56, wherein the oligomer sequence is 5′-AGCATTA-3′ (SEQ ID NO:987).
 59. The oligomer according to claim 45, wherein the contiguousnucleotide sequence of the oligomer is 8 nucleotide units in length, allthe nucleotide units are LNA units, all the internucleoside linkages arephosphorothioate, and all cytosine LNA units are 5-methylcytosines. 60.The oligomer according to claim 59, wherein the oligomer sequencecomprises 5′-TAGCATTA-3′ (SEQ ID NO: 4).
 61. The oligomer according toclaim 59, wherein the oligomer sequence is 5′-TAGCATTA-3′ (SEQ ID NO:4).
 62. The oligomer according to claim 45, wherein the contiguousnucleotide sequence of the oligomer is 9 nucleotide units in length, allthe nucleotide units are LNA units, all the internucleoside linkages arephosphorothioate, and all cytosine LNA units are 5-methylcytosines. 63.The oligomer according to claim 62, wherein the oligomer sequencecomprises 5′-TTAGCATTA-3′ (SEQ ID NO: 986).
 64. The oligomer accordingto claim 62, wherein the oligomer sequence is 5′-TTAGCATTA-3′ (SEQ IDNO: 986).
 65. The method according to claim 39, wherein the disease ormedical disorder is cancer.
 66. The oligomer according to claim 35,wherein the oligomer is conjugated with at least one non-nucleotide ornon-polynucleotide moiety.
 67. The oligomer according to claim 66,wherein the non-nucleotide or non-polynucleotide moiety is selected froma protein, a fatty acid chain, a sugar residue, a glycoprotein, apolymer, or any combination thereof.
 68. The oligomer according to claim67, wherein the protein is an antibody.
 69. The oligomer according toclaim 67, wherein the polymer is polyethylene glycol.
 70. The oligomeraccording to claim 35, wherein at least one of the internucleosidelinkages present between the nucleotide units of the contiguousnucleotide sequence is not a phosphorothioate or a phosphodiesterinternucleoside linkage.
 71. The oligomer according to claim 35, whereinat least one cytosine LNA units is not 5-methylcytosine.
 72. Theoligomer according to claim 35, wherein at least one cytosine LNA unitis 5-methylcytosine.
 73. The oligomer according to claim 35, wherein allcytosine LNA units are 5-methylcytosines.
 74. The oligomer according toclaim 40, wherein at least one cytosine LNA units is not5-methylcytosine.
 75. The oligomer according to claim 40, wherein atleast one cytosine LNA unit is 5-methylcytosine.
 76. The oligomeraccording to claim 40, wherein all cytosine LNA units are5-methylcytosines.
 77. A method for the treatment of a disease ormedical disorder associated with the presence or over-expression of amicroRNA, comprising administering an oligomer to a patient who issuffering or likely to suffer from said disease or medical disorder,wherein the oligomer comprises a contiguous sequence of 7 to 11nucleotides units in length, wherein at least 70% of the nucleotideunits of the oligomer are selected from the group consisting of LNA(Locked Nucleic Acid) units and 2′ substituted nucleotide analogues,wherein at least 50% of the nucleotide units of the oligomer are LNAunits, and wherein at least one of the internucleoside linkages presentbetween the nucleotide units of the contiguous nucleotide sequence is aphosphorothioate internucleoside linkage.